Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.