Paromomycin is an antileishmanial chemotherapeutic agent. Leishmania donovani promastigotes resistant to 800 microM of paromomycin were selected by increasing drug pressure and cloned. These promastigotes did not acquire multidrug resistance. Paromomycin resistance was stable in the absence of the drug in the culture. It remained stable also in amastigotes isolated after a passage in mice. Furthermore the resistant parasites were still infective to macrophages in vitro and for mice in vivo. A sensitive method to detect and to quantify intracellular paromomycin by HPLC was developed and allowed to show that the main mechanism of resistance seems to be due to decreased drug uptake probably as a consequence of altered membrane composition.