The pLEX series of vectors was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. These pUC-based constructs contain one of three independent selectable markers and a multiple cloning site inserted between the upstream and downstream untranslated regions of the previously cloned Leishmania major HEXBP gene. Selection was based on resistance to the aminoglycosides, hygromycin B and neomycin, and to nourseothricin, a novel independent selectable marker for transfection of Leishmania. The vectors were introduced into Leishmania promastigotes by electroporation and were maintained as extrachromosomal circular concatemers containing between four and eight repeat units of the pLEX monomer. To demonstrate the efficient expression of cloned exogenous genes using the pLEX system, promastigotes were transfected with a pLEX construct that contained a second drug-resistant selectable marker gene cloned into the expression site, and clones were obtained that grew on media containing two antibiotics. These vectors, together with the novel selectable marker, will further facilitate the molecular analysis of gene expression in Leishmania.