Drug sensitivity of clinically antimony-unresponsive Leishmania donovani isolates from Eastern Sudan was evaluated in an in vitro culture system against sodium stibogluconate (Pentostam) and Amphotericin B. Eight isolates, six from antimony-resistant and two from clinically responsive patients were included in the study. Parasites were tested as promastigotes and four of them were selected to be tested as amastigotes using a murine macrophage-like cell line. The results indicated that the conventional promastigotes and amastigotes-screening assays did not correlate with the clinical picture of patients. In vivo unresponsiveness does not necessarily mean primary parasite resistance. Amphotericin B could be a suitable second line drug in patients unresponsive to pentostam and without concomitant diseases, if close hospital monitoring is available. Promastigotes sensitivity testing concentrations are virtually incomparable with the in vivo clinically curable doses and the amastigotes/macrophage test concentrations.