We have found that the hemolytic potency of P30 - a subcellular fraction that contains most of the hemolytic activity of Entamoeba histolytica trophozoites--increased up to 100 times by preincubation for 36 h at 36 degrees C. This remarkable increase in potency was due to the in vitro generation of heat-labile as well as heat-stable hemolytic components and could be prevented if P30 was preincubated at 4 degrees C or subjected to either heating at 90 degrees C or to repeated freeze-thawing cycles before preincubation at 36 degrees C. The major hemolytic activity of E. histolytica thus appears to depend on latent, heat-labile and heat-stable hemolysins that are probably unmasked through the action of endogenous amebal enzymes.