Trypanosoma brucei (TB) cultured in rat blood, bovine serum or lipid-depleted serum generated distinct differences in cholesterol availability. Whereas cell proliferation of the parasite was relatively unaffected by cholesterol availability, the ratios of cellular ergostenols to cholesterol varied from close to unity to three orders of magnitude different with cholesterol the major sterol (> 99%) of bloodstream form cells. In the procyclic form cultured with lipid-depleted serum, 15 sterols at 52 fg/cell were identified by GC-MS. The structure of these sterols reveal a non-conventional ergosterol pathway consistent with the novel product diversity catalyzed by the recently cloned sterol methyltransferase (SMT). A potent transition state analog of the TB SMT C24-alkylation reaction, 25-azalanosterol [Ki 39 nM], was found to inhibit the growth of the procyclic and bloodstream forms at an IC50 of ca. 1 uM. This previously unrecognized catalyst-specific inhibition of cell growth was unmasked further using 25-azalanosterol-treated procyclic form which, compared to control cultures, causes a change in cellular sterol content from ergostenols to cholesterol. However, growth of the blood stream form disrupted by 25-azalanosterol was not rescued by cholesterol absorption from the host uggesting an essential role for ergosterol (24-methyl sterol) in cell proliferation and that SMT can be a new enzyme target for drug design.