The fine structural localization of enzyme activity in the trematode Haematoloechus medioplexus has provided a greater discrimination of enzyme localization previously studied by light microscopy. Several phosphate-bearing compounds were utilized as substrates in alkaline and acid media to detect alkaline and acid phosphatases, with lead as the trapping agent. Lead was also used as a trapping agent with thiolacetate, to localize another esterase beside phosphatase. The only phosphatecontaining substrate to be hydrolyzed in alkaline media was adenosine triphosphate, with localization on the outer cuticular membrane and in the spines. Two different acid phosphatases may have been present. One acted on phenyl phosphate with a distribution in the cuticle. The other acted on glyceraldehyde-3-phosphate with a distribution in the cuticle and spine. A nonspecific esterase reacting with thiolacetate was found in the cuticle and in the spines. Colloidal ferritin and colloidal thorium dioxide were used to localize macromolecular transport and to determine its mechanism. The major colloid transport was found to be through the cuticle by transmembranosis. Colloid was also found in the spine. Light microscope studies on histochemical enzyme localization in trematodes have been reviewed by Lee (1967), and Halton (1967). The present report describes an electron microscope study of the fine structural localization of enzyme activity in the absorbing surfaces of the trematode Haematoloechus medioplexlts. Colloidal transport in H. medioplexus is also considered. MATERIALS AND METHODS The trematode was dissected from the lungs of the grass frog, Rana pipiens, in Krebs-Ringer tris maleate solution, pH 7.4 (KRTM). The worms were rinsed thrice in KRTM, carefully removing adhering debris. One group of worms was set aside for the colloid transport experiment. The other group was fixed in ice-cold 4% formaldehyde (made with paraformaldehyde) prepared in 0.1 M cacodylate buffer, pH 7.4. After 60 min in the cold fixative the worms were rinsed six times in cold buffer, 10 min each rinse. A portion of fixed worms was taken as controls for the colloid transport experiment. The rest of the fixed and washed worms were sectioned at 250 g and incubated in one of the enzyme localization test solutions (see below) at room temperature (23 to 26 C) for 30 min. The experimental lead solution for the phosphatases was that of Wachstein and Meisel (1957) and the lead method for esterase was that of Barrnett (1962). The substrates for localizing alkaline phosphatase at pH 7.4 were present in the test solution at 1 mM and included adenosine triphosphate Received for publication 27 November 1967. * Supported by a research grant, AI-06442, from the NIH. (ATP), glucose-6-phosphate, fructose diphosphate, phenylphosphate (4P), glyceraldehyde-3-phosphate (G3P), and adenosine diphosphate. The substrates at 1 mxl for detecting acid phosphatase at pH 5.0 included G3P and ,P. Thiolacetate was the substrate for determining the distribution of esterase activity, other than phosphatase. The inhibitors tested at 1 mM included: Nethyl maleimide (NEM), 2,4, dinitrofluorbenzene (DNFB), p-chloromercuribenzoic acid (PCMB), cholic acid, ouabain (using tris-HCl buffer rather than KRTM), and eserine sulfate. An incubating medium at pH 7.4 and at pH 5.0, with the substrate omitted, was run as a control for the enzyme reactions. Following incubation in the control and experimental solutions, the sectioned portions of the worms were rinsed three times with cold buffer. Several sections of each of the experimental and control trials with phosphate substrates were treated with 1% ammonium sulfide, to clarify the sites of phosphatase activity for preliminary light microscope observations of results and enzyme localization. The precipitate formed by the degradation of thiolacetate in the presence of lead permitted preliminary light microscope observations of results and enzyme localization without further treatment of the incubated test and control sections. The sections used for preliminary observation were saved and not prepared for electron microscopy. To study colloid transport the unfixed and fixed entire trematodes were incubated in either a 2% suspension of ferritin or a 2% suspension of ThO2 (as Thorotrast) in KRTM at pH 7.4 for 120 min at room temperature (23 to 26 C). Following the exposure to colloid the whole worms were rinsed in three changes of KRTM, gently agitated in each change to remove extraneous colloid and placed in ice-cold cacodylate buffered 3% glutaraldehyde for 120 min. The worms