The antiprotozoal activity of chlorpromazine against the pathogenic protozoan Leishmania donovani, in both its amastigote and promastigote stages, was characterized. Chlorpromazine at concentrations greater than or equal to 3.12 micrograms/ml (9.8 X 10(-6) M) produced a significant reduction in viable promastigotes. The minimal protozoacidal concentration for promastigotes, defined as that concentration which produced greater than or equal to 90% reduction in viable parasites after 18 h, was 13.8 micrograms/ml. The results were similar when promastigote viability was assessed by flagellar motility or by the ability of drug-exposed or control promastigotes to incorporate [3H]uridine and [3H]leucine. Exposure of promastigotes to 50 micrograms of chlorpromazine per ml reduced O2 consumption by 87% within 30 min and immobilized 97% of parasites. Morphological disruption of promastigotes was observed by electron microscopy. The mean minimal protozoacidal concentration of chlorpromazine for amastigotes was 13.2 micrograms/ml. Chlorpromazine given orally (20 mg/kg per day for 14 days) reduced the parasite burden in L. donovani-infected hamsters by 64.2% (P less than 0.01) as measured by the number of amastigotes in touch preparations of livers and by 67.9% (P = 0.03) as measured by the number of promastigotes derived from homogenates of spleens. This dose is ca. 10-fold greater than that tolerated by patients being treated for psychiatric illness. Although chlorpromazine will probably not be useful in the treatment of human visceral leishmaniasis, the data suggest that less-toxic phenothiazines might prove to be effective.