1. Two partially purified acid phosphatase activities present in American Leishmania promastigote homogenates were characterized by biochemical methods. 2. One isoenzyme acts preferentially on p-nitrophenyl phosphate, is strongly inhibited by 30 mM alloxan, citrate, maleate, malonate and succinate, and strongly stimulated by 3 mM spermine. Its pI is 4.8. 3. The other isoenzyme acts preferentially on beta-glycerophosphate and is resistant to 30 mM alloxan, citrate, maleate, malonate and succinate and also to 3 mM spermine. Its pI is 5.7. 4. Both acid phosphatase isoenzymes have an optimum pH of 5.2, are tartrate-sensitive and strongly membrane-bound, as shown by differential centrifugation and density gradient equilibration. 5. Both isoenzymes were separated by using homogenates prepared in 2% Triton X-100, differential centrifugation, Sepharose 4B/CL-4B gel filtration, ion exchange chromatography and electrofocusing. After this procedure, they were still contaminated with several different proteins. 6. Purification was around 150-fold, with a 32% yield. 7. When these acid phosphatase activities were measured in total homogenates from 12 different Leishmania isolates, p-nitrophenyl phosphatase specific activity values were quite close; beta-glycerophosphatase-specific activity had around a 2-fold variation. 8. This variation was independent from taxonomic classification or infectivity of susceptible hosts.