Protozoan parasites of the genus Leishmania synthesise lipophosphoglycans, phosphoglycans and proteophosphoglycans that contain phosphosaccharide-repeat units of [-6Galß1-4Mana1-P-]. In this study, a GDP-Man-dependent a-mannosylphosphate-transferase activity was detected in washed Leishmania major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. The divalent-cation-dependent a-mannosylphosphate-transferase activity had an apparent Km for GDP-Man of about 15-20µM and a pH optimum of 7.0. The activity showed a requirement for a nonreducing terminal ßGal residue and for one or more phosphodiester units preceding the acceptor site. Based on these results, the activity may be defined as a GDP-Man: Galß1-4Mana1-P-R a-mannosylphosphate-transferase. This acceptor specificity is consistent with a role for the a-mannosylphosphate transferase in the elongation of phosphosaccharide-repeat domains of Leishmania glycoconjugates rather than in the priming of these domains. An identical or similar activity must exist in the amastigote forms of the Leishmania that produce and secrete proteophosphoglycan material and the activity therefore represents a feasible target for the development of chemotherapeutics.