Standard courses of pentavalent antimonials frequently fail to cure cutaneous, mucocutaneous, and visceral leishmaniasis, and characteristically fail to cure diffuse cutaneous We have determined the in vitro sensitivity of clinical isolates of Leishmania to pentavalent antimony to determine if inherent drug resistance of the parasite is responsible for treatment failures in human beings. Intracellular amastigotes resulting from promastigote-initiated infection of human macrophages were exposed to pentavalent antimony for 6 days at 34.5–35°C. Amastigotes from clinically sensitive simple cutaneous lesions exhibited a range of in vitro sensitivity. Four strains were ≥90% eliminated and two strains were 70–75% eliminated in vitro by concentrations of antimony (15–20 µg Sb/ml), comparable to peak achievable serum levels in humans. Amastigotes from initially clinically resistant simple cutaneous lesions showed a wider range of sensitivities. Five strains were ≥90% eliminated, but one strain was only 40% eliminated and another strain was completely insensitive in vitro. The clinically resistant diffuse cutaneous strain was 61% eliminated. The techniques described herein permit determination of the in vitro antimicrobial susceptibility of Leishmania from all major human forms of leishmaniasis. The data from this series indicate that in a minority of initially resistant cases, parasite resistance to the drug may be contributing to clinical resistance, and use of non-antimonial drugs might be recommended for future therapy.