Conditional transgene expression is a potentially useful approach to investigate complex biological systems in vivo. We recently demonstrated that tetracycline-responsive promoters could be employed to achieve regulated, cardiac-specific expression of target genes in transgenic mice. To more fully define the quantitative and spatial parameters associated with tetracycline-regulated gene expression in the heart, we crossed transgenic mice harboring either a firefly luciferase or a nuclear-localized bacterial lacZ target gene with strains expressing a tetracycline-controlled transactivator (tTA) under the regulatory control of 2.9 kb of 5′ flanking sequence from the rat α-myosin heavy chain gene. Luciferase activity was induced nearly 300-fold in the hearts of binary-transgenic mice compared with mice carrying only the luciferase reporter gene. No significant transactivation was observed in any other tissues examined. Binary transgenics harboring the lacZ reporter gene showed substantial β-galactosidase activity throughout the heart, but the response of individual cardiac myocytes was heterogeneous. For both reporter genes, tetracycline treatment fully repressed tTA-dependent transactivation. These data provide important insights into the nature of studies that can be successfully addressed using the tetracycline-regulated gene expression system in the heart.