Infective stages of the protozoan parasite Leishmania spp. accumulate a class of beta-1,2-mannan oligosaccharides as their major carbohydrate reserve material. Here, we describe the biosynthesis of Leishmania mannan. Mannan precursors were identified by metabolic labeling of Leishmania mexicana promastigotes with [(3)H]mannose. Label was initially incorporated into a phosphomannose primer and short phosphorylated beta-1,2-mannan oligomers that were two to five residues long. Analysis of the mannan primer by Fourier transform ion-cyclotron resonance MS and various enzymatic and chemical treatments and comparison with authentic mannose (Man) phosphates indicated the presence of Man-alpha-1,4-cyclic phosphate. This primer was synthesized from Man-6-phosphate by means of Man-1-phosphate in a cell-free system. Short mannan chains containing the primer were subsequently dephosphorylated and then further elongated by GDP-Man-dependent transferases in vivo and in the cell-free system. The synthesis of this glycan primer likely constitutes a key regulatory step in mannan biosynthesis and is a potential target for antileishmanial drugs.