Proteolytic activities of the protozoan parasite Entamoeba histolytica strain HM1:IMSS and the cytochalasin D-resistant mutant BG-3 were analyzed following stimulation with collagen type I and Ca2+, which induces electron-dense associated collagenase secretion. The mutant BG-3 had a protease activity of 73 kDa and secretion of total protease activity was not stimulated by collagen type I and Ca2+, which produced, in contrast, a 2-fold increase in protease secretion by the parental strain. This collagen-stimulated protease secretion was inhibited by cytochalasin D at a concentration of 1 microgram/ml. Cytochalasin D did not have any effect on the protease activity released by the mutant BG-3. These findings suggest that cytoskeleton integrity is necessary for collagen-induced protease secretion.