Histochemical studies, using the light and electron microscopes, have been made of carboxylic esterase activity in the tegumental cells and surface tegument of the frog lung fluke Haplometra cylindracea. Regional differences in reactivity for esterases were found. The tegumental cells and tegumental lining of the two suckers and tegumental cells connected to the tegument surrounding the oral sucker stained intensely for esterases, but the tegumental cells and associated tegument in the rest of the body stained only weakly or not at all. The use of specific substrates or eserine sulfate showed that most of the activity was due to acetylcholinesterase. With the electron microscope, enzyme reaction product was found at the nuclear membranes and endoplasmic reticulum of esterase-positive tegumental cells and, in large concentrations, on the membranes of numerous platelike inclusion bodies present in the cells and connecting tegument. No structural differences relating to enzyme activity were found between reactive and nonreactive tegumental cells and tegument. The acidophilic cells present in the oral region of the worm were at all times negative for esterase activity. The oral region of the digenetic frog lung fluke, Haplometra cylindracea, is known to be a site of fairly intense carboxylic esterase activity (Halton, 1967a). Nonspecific esterases have been demonstrated using light microscopy in the oral and ventral suckers and their tegumental lining, as well as in certain gland cells which lie immediately behind the oral sucker. Ductlike processes connect these cells to the tegument overlying the oral sucker, and their content and that of the associated tegument is esterase-positive. Electron microscope observations have shown that at least two cell types are in communication with the tegument in H. cylindracea. There are numerous tegumental cells with tubelike connections to the surface tegument or cuticle (Threadgold, 1968) and, in the anterior region, fewer, but somewhat larger, acidophilic cells which are drawn out into Received for publication 7 July 1968. ductlike structures that open to the exterior through pores in the tegument (Halton and Dermott, 1967). The present investigation was made in order to correlate these light and electron microscope observations and to determine the source of carboxylic esterase activity in this region of the worm. MATERIALS AND METHODS For light microscopy live specimens of H. cylindracea were removed from the lungs of locally caught frogs (Rana temporaria) and fixed for 3 hr at 4 C in 10% formalin, buffered to pH 7.2 with 0.2 M phosphate. The specimens were then washed in three 10-min changes of ice-cold phosphate buffer, frozen in 5% sucrose solution, and sectioned. Sections (10 ,u thick) were transferred to glass slides and allowed to dry in air (30 min) prior to incubation. Carboxylic esterase activity was demonstrated using the standard techniques described in Pearse (1960). The following incubation times and temperatures were used: 12 hr at 18 C for 5-bromoindoxyl acetate (5-BIA); 12 hr at 18 C for acetyl FIGURES 1-3. Light histochemical localization of carboxylic esterase activity in H. cylindracea. 1. Frontal section through oral region showing esterase activity in oral sucker and tegumental lining and in tegumental cells and connecting tegument overlying oral sucker. 5-BIA. X 140. 2. Sagittal section through part of oral sucker showing intense esterase activity in tegumental cells of the sucker, their connections to the tegument lining the sucker, and throughout the tegument itself. Note esterase-positive connections from cells behind oral sucker to tegument overlying the sucker. 5-BIA. X 210. 3. Section through esterase-positive tegumental cells located immediately behind oral sucker. These connect by tubelike processes to the tegument overlying oral sucker and shown in Figure 2. 5-BIA. X 350. Abbreviations: A.C., acidophilic cell; C., connection; G.E.R., granular endoplasmic reticulum; I.B., inclusion bodies; M., muscle; N., nucleus; Nu., nucleolus; O.S., oral sucker; Ph., pharynx; P.M., plasma membrane; S.T., sucker tegument; T., tegument; T.C., tegumental cell.