We report here a second-generation tetracycline-responsive repressor-operator system in Leishmania donovani. In this system, expression of a reporter luciferase gene (LUC) is driven by the inducible Leishmania ribosomal RNA promoter on the DNA strand opposite to a hygromycin resistance gene (HYG) whose expression is driven by the endogenous pol I promoter on chromosome 27 (rDNA locus) or the endogenous pol II promoter on chromosome 35 (LD1 locus). Transgenic cell lines showed regulation of LUC gene expression over three orders of magnitude. In the absence of tetracycline, luciferase expression levels were 2-3-fold higher than machine background when integrated into the LD1 locus, but was over 10-fold higher than machine background when integrated into the rDNA locus.