Promastigotes resistant to sinefungin (SF), a nucleoside antibiotic that is structurally related to S-adenosylmethionine (AdoMet), were obtained starting from two cloned strains of Leishmania donovani. The resistance was induced by increasing the drug pressure gradually until promastigotes capable of growing in the presence of concentrations 10,000 times higher than the 50% growth-inhibitory (IC50) values for the control cells were obtained. The resistance to SF of both clones was specific and stable in the absence of drug pressure. High-performance liquid chromatographic (HPLC) analyses indicated highly reduced levels of SF in the two resistant clones. However, the intracellular SF concentration in these resistant cells was much higher than the IC50 values for wild-type cells. In one clone, the decreased drug uptake was coupled to a decrease in the affinity of two protein methylases for SF, whereas in the other clone the biosynthesis of polyamine precursors was modified. This study demonstrates that resistance to a drug molecule with pleiotropic targets can be developed through various mechanisms by different strains.