Tetracycline- and ecdysone-responsive systems have recently been developed for regulated gene expression in eukaryotic cells. Using nucleolar-targeted luciferase and beta-galactosidase as reporter proteins we demonstrate that both systems can function simultaneously and independently in a continuous human cell line. Both gene activities could be regulated over a broad range and at the single cell level by the concentrations of tetracycline and muristerone A, respectively, in the culture medium. The strategy described here will allow to investigate the function and interaction of two separate gene activities in a well-defined and reproducible cellular context.