Antimonial-containing drugs are the first line of treatment against the parasite Leishmania. Resistance to antimonials has been correlated to its reduced accumulation. We used a dominant negative functional cloning strategy where a Leishmania mexicana expression cosmid bank was transfected in cells resistant to trivalent antimony (SbIII). Cells were selected for increased sensitivity to SbIII. One cosmid was isolated that could bestow SbIII sensitivity to resistant cells. The gene part of this cosmid that is responsible for increased SbIII sensitivity corresponds to AQP1, an aquaglyceroporin. AQP1 was recently shown to be a route by which SbIII can accumulate in Leishmania cells. Transport studies have shown that the L. mexicana AQP1 can restore SbIII transport in resistant cells. Southern blot analysis indicated that the copy number of neither the AQP1 gene nor the other AQP homologues was changed in antimony-resistant mutants of several Leishmania species. The AQP1 gene sequence was also unchanged in mutants. However, the AQP1 RNA levels were downregulated in several Leishmania promastigote species resistant to antimonials. In general, but not always, the level of AQP1 transcript levels correlated well with the accumulation of SbIII and resistance levels in Leishmania cells. AQP1 thus appears to be a key determinant of antimonials accumulation and susceptibility in Leishmania.