The ability to specifically and efficiently express selected genes in tumor cells is an important goal for cancer gene therapy. Transcriptional targeting of adenovirus to tumor cells, thereby limiting their expression to specific cell types, represents one experimental approach to this problem. We have previously shown that a recombinant adenovirus containing the murine tyrosinase promoter coupled to a dimer of the tyrosinase-enhancer element can target the expression of beta-galactosidase cDNA to melanoma cells. We now report that this same promoter/enhancer cassette can efficiently drive the expression of the herpes simplex virus thymidine kinase gene in melanoma cells. Infection of melanoma cells with the AdmTyr-tk virus along with subsequent ganciclovir treatment induces S phase cell cycle arrest associated with a profound change in cell size and morphology. Treated cells remain viable for prolonged periods, but clonogenic assays demonstrate that the cell cycle arrest is irreversible. In contrast, nonmelanoma cells are unaffected by this treatment regimen, exhibiting normal growth kinetics, metabolic activity, and cell cycle progression. The therapeutic efficacy of the AdmTyr-tk virus was tested in vivo using a xenograft model of human melanoma. The injection of the AdmTyr-tk virus into established subcutaneous tumor nodules in combination with systemic ganciclovir administration led to a decreased tumor growth rate and to complete tumor regressions in some cases. These studies demonstrate the feasibility of selectively targeting growth-inhibitory genes to melanoma cells in vitro and in vivo.