Hamycin, a polyene antibiotic related to amphotericin B, has been used topically to treat fungal and protozoan infections in India. We assessed the cytotoxic activity of hamycin on nine metronidazole resistant or susceptible Trichomonas vaginalis strains isolated from symptomatic or asymptomatic patients. Cytotoxic activity of hamycin against two mammalian cell lines, BHK-21 and HeLa, was also determined. Tritiated thymidine pulse labeling after drug-washout and a recovery period was used to distinguish death of target cells from temporary static effects of the drug. Liposomal hamycin and hamycin formulated with dimethyl sulphoxide, deoxycholate or glycerin were compared. Although all hamycin preparations were trichomonacidal at approximately 1 mg/L, hamycin-dimethyl sulphoxide was stable for only 24 h, and hamycin glycerin was incompletely solubilized. Hamycin-deoxycholate remained a stable gel for 2 months at room temperature, but its activity was reduced four-fold when compared to the fresh preparation. The mammalian tissue culture cell lines HeLa and BHK-21 were killed by trichomonacidal concentrations of hamycin-deoxycholate. This cytotoxicity of hamycin for mammalian cells is a concern, but new forms of the drug are under development that may allow more widespread use of this drug.