Lipophosphoglycan (LPG) is the predominant surface glycoconjugate of Leishmania promastigotes and plays several roles in the infectious cycle of this protozoan parasite. The salient feature of LPG is the presence of 15-30 copies of a disaccharide-phosphate repeating unit Gal(beta1,4)Man(alpha1-PO4), which is also found on many other secreted molecules (secretory acid phosphatase, phosphoglycan, proteophosphoglycan). This structural diversity suggests that a multiplicity of enzymes mediating repeating unit addition may exist, especially for the mannosylphosphoryltransferases (MPTs), which initiate repeating unit synthesis. This work has taken a combined biochemical-genetic approach to resolve this issue. An lpg- mutant of Leishmania donovani, JEDI, was obtained by antibody selection against cells expressing a repeating unit epitope of LPG. Metabolic and surface labeling experiments revealed that JEDI cells accumulated a truncated form of LPG bearing only a single repeating unit: [Gal(beta 1,4)Man(alpha1-PO4)][Gal(alpha1,6)Gal(alpha1,3)Gal(f)(beta1,3)[Glc(alpha 1-PO4)]Man(alpha1,3)Man(alpha1,4)GlcN(alpha1,6)]-PI. Enzymatic assays of microsomal preparations showed that JEDI lacked MPT activity when tested with a repeating unit acceptor but retained wild-type levels of the MPT activity with an LPG glycan core acceptor. These data indicate that at least two distinct MPT activities are required for LPG repeating unit synthesis: one involved in the 'initiation' of repeating unit synthesis on the LPG core (iMPT), and a second (lacking in JEDI) participating in the 'elongation' phase of repeating unit addition (eMPT), leading to the mature full-length LPG.