Hymenolepis diminuta contains an enzyme hydrolyzing acetylcholine at a high rate. It appears to be specific for this substrate by criteria used in previous studies on other organisms. Most of the activity is in a particulate fraction. Acetylcholinesterase activity was inhibited by atropine sulfate and eserine sulfate but was not inhibited by diisopropylfluorophosphate. Specific acetylcholinesterase has been demonstrated in the trematodes, Schistosoma mansoni (Bueding, 1952) and Fasciola hepatica (Chance and Mansour, 1953; Frady and Knapp, 1967), and in the nematodes, Litomosoides carinii, Ascaris lumbricoides (Bueding, 1952), and Haemonchus contortus (Lee and Hodsden, 1963). It probably occurs in Echinococcus (Schwabe et al., 1961). Cholinesterases of undefined specificity have been reported from a number of other helminths (reviewed by von Brand, 1966). Although cholinesterase has been demonstrated in Hymenolepis diminuta (Lee et al., 1963; Schardein and Waitz, 1965), the only demonstration that the worm has an enzyme with high specific affinity for acetylcholine is histochemical (Wilson, 1965). The present experiments were designed to further examine the specificity of the enzyme. MATERIALS AND METHODS H. diminuta was reared in young male SpragueDawley rats and harvested 14 to 20 days after infecting the hosts. The worms were flushed from the gut and washed in six changes of ice-cold buffered Ringer (pH 7.4 with 0.025 M tris-maleate buffer). All subsequent operations up to the incubations were carried out at 0 to 4 C. The worms were homogenized in a glass homogenizer containing 2 ml of saline (0.15 M NaCl, 0.04 M MgCl2, and 0.25 M NaHCO3) for each 50 mg of tissue. Fractionation of homogenates was carried out in an International centrifuge with a high-speed attachment. Dry weights of duplicate aliquots of homogenates or homogenate fractions were determined after heating to constant weight at 105 C. Acetylcholinesterase activity was measured by the method of Nachmansohn and Rothenberg (1945), in a conventional Warburg apparatus under an atmosphere of 95% N2-5% CO2 at 37 C. After a Received for publication 26 June 1967. * Supported in part by a grant from the NIH (Al 01384). t Present address: Weber State College, Ogden, Utah. 15-min gas and temperature equilibration, the substrate was added from the side arm of the Warburg vessel into the main compartment in a total reaction volume of 2.5 ml. Enzyme activity was recorded as microliters CO2 evolved per mg dry weight during the first 15 min. Control vessels lacking homogenate or substrate were run to make corrections for nonspecific gas evolution. Unless otherwise indicated, the substrate concentration was 40 msM. RESULTS AND DISCUSSION The data summarized in Table I and Figure 1 indicate the presence in H. diminuta of a specific acetylcholinesterase by the same criteria used in previous studies on vertebrate and helminth tissues: 1) in concentrations at which acetylcholine is hydrolyzed at a high rate butyrylcholine is not hydrolyzed as well, 2) enzyme activity is reduced when the substrate concentration is above or below an optimum, and 3) triacetin is hydrolyzed at a slow