A mutant cell line of Leishmania donovani (R2D2), previously selected for resistance to the cytotoxic lectin ricin agglutinin, was found to be totally deficient in the synthesis and expression of lipophosphoglycan, a dominant surface virulence factor. The metabolic defect in R2D2 parasites responsible for its lipophosphoglycan (LPG-) phenotype was investigated in this study. Following metabolic labeling of R2D2 parasites with either [3H]galactose or [3H]mannose, the main glycosylphosphatidylinositide product that accumulated was Glc-PO4-Man-Man-GlcN-lyso-1-O-alkylphosphatidylinositol (PI). The metabolic defect was further defined using a cell-free glycosylation system. When membrane preparations from wild-type cells were incubated with UDP-[3H]galactose and unlabeled GDP-mannose in the absence of exogenous acceptors, radiolabeled lipophosphoglycan was synthesized. The addition of exogenous Man-Man-GlcN-PI or Galf-Man-Man-GN-PI stimulated lipophosphoglycan synthesis in vitro. In contrast, when membrane preparations from R2D2 cells were incubated with exogenous Man-Man-GlcN-PI as an acceptor or in the absence of exogenous acceptor, the truncated glycosylphosphatidylinositide Glc-PO4-Man-Man-GlcN-PI was the main radioactive product synthesized. However, when exogenous Galf-Man-Man-GN-PI was added to the R2D2 in vitro system, radioactive lipophosphoglycan was synthesized. Collectively, these results indicate that the mutant R2D2 cells are unable to complete the assembly of the glycan core of LPG because of a defect in the synthesis of the "activated" galactofuranosyl donor or the lack of a functional galactofuranosyltransferase.