Methotrexate (Mtx) conjugated with maleylated bovine serum albumin (Mtx-MBSA) was taken up and degraded by cultured hamster peritoneal macrophages through the polyanion binding site for acetylated low density lipoprotein. Mtx-MBSA also eliminated intracellular amastigotes of Leishmania donovani in cultured hamster peritoneal macrophages about three times more efficiently than free Mtx. The antileishmanial effect of Mtx-MBSA on parasitized macrophages was blocked by MBSA, lysosomal inhibitors (chloroquine and monensin), and metabolic antagonists of Mtx (folic and folinic acids). The primary sites of accumulation of radioactivity of injected 125I-labeled Mtx-MBSA were the macrophage rich tissues, viz. liver and spleen. These results suggest that Mtx-MBSA would ensure rapid and effective killing of intracellular parasites harbored by macrophages. Furthermore, these results also indicate the feasibility of a general approach for rapid intracellular delivery of desired agents to macrophages for various purposes.