We report that in vitro sensitivity to pentavalent antimony (Sb5) of 35 Leishmania isolates as determined by the semiautomated microdilution technique (SAMT) showed an 89% and 86% correlation with clinical outcome after Pentostam and Glucantime treatment, respectively. These results suggest that in over 85% of the cases, the clinical outcome of treatment (cure or failure) could have been predicted by using the SAMT technique. Furthermore, the results clearly indicate that drug resistance is a problem, and that at least in some instances, failure to respond to treatment is due to the parasite as well as patient factors. Strains from Sb5-treated patients with American cutaneous and mucocutaneous disease who fail at least one complete course of Pentostam are as highly nonresponsive to this drug as laboratory-proven drug-resistant Leishmania strains. It was determined that some Leishmania isolates are innately less susceptible to Sb5 than others, and that moderate resistance to Sb5 exists in nature. A 10- and 17-fold increase was detected in the 50% inhibitory concentration (IC50) of Sb5 for L. mexicana and L. braziliensis isolates after subcurative treatment of the patients, when compared with the mean IC50 of seven and six isolates from the same endemic areas in Guatemala and Peru, respectively. Thus, we have correlated subcurative treatment to a decrease in drug sensitivity in at least these two cases. Collectively, these results indicate that under Sb5 pressure from undermedication, the parasites inherently most drug resistant are favored. The degree of resistance of a strain to antimony in association with host-specific factors will determine whether the clinical response to treatment with this drug is a total cure or a partial response followed by relapse(s), and possibly secondary unresponsiveness resulting in total resistance to antimony. It is evident from our in vitro test data that the SAMT is an extremely powerful and highly accurate technique for the prediction and determination of drug sensitivity of leishmanial isolates, as well as a means to screen for anti-leishmanial agents.