Homogenates of Giardia lamblia trophozoites exhibited the following hydrolase activities: acid phosphatase (EC 126.96.36.199), proteinase (EC 3.1.4) with urea-denatured hemoglobin and N-benzoyl-DL-arginine-2-naphthylamide as substrates, deoxyribonuclease (EC 188.8.131.52), and ribonuclease (EC 184.108.40.206). beta-N-Acetylglucosaminidase (EC 220.127.116.11), beta-galactosidase (EC 18.104.22.168), beta-glucuronidase (EC 22.214.171.124), alpha-D-glucosidase (EC 126.96.36.199), beta-D-glucosidase (EC 188.8.131.52), and beta-D-xylosidase (EC 184.108.40.206) activities were below the level of detection. Differential and isopycnic centrifugation of homogenates demonstrated that giardial hydrolases were localized in a single-particle population sedimenting at 7200g for 30 min. The particles had a buoyant density in sucrose of 1.15 and exhibited latency. Latency was completely destroyed by Triton X-100 or 15 cycles of freezing and thawing. After centrifugation of Triton- or freeze-thaw-treated particle fractions, the hydrolase activities, though no longer latent, were still sedimentable suggesting tight binding to the organelle membrane. Latency was destroyed simultaneously for all hydrolases, in direct proportion to the amount of Triton added to a particle preparation or to the number of times a particle preparation was subjected to freezing and thawing. These results support the suggestion that the hydrolases of G. lamblia trophozoites are localized in a single-particle population of lysosome-like organelles.