One potential avenue for future cancer therapy involves the specific targeting of effector genes to cancer cells throughout the body, including distant metastatic sites. As a first step toward this goal, we tested the ability of the transcriptional regulatory elements of the human and mouse tyrosinase genes to promote high levels of pigment cell-specific transcription. A construct consisting of 209 bp of the human tyrosinase promoter linked to two enhancer elements was demonstrated to drive high-level, melanoma-specific expression of a beta-galactosidase (beta-gal) reporter gene in transient transfection assays. In studies of the murine tyrosinase promoter region, constructs containing up to 2500 bp of the 5' regulatory region were found to have very low transcriptional activity in murine melanoma cells. However, as with the human system, addition of two tandem repeats of an upstream enhancer element resulted in high levels of lineage-specific transcriptional activation. The murine tyrosinase promoter-enhancer expression cassette was introduced into the E1 region of a recombinant adenovirus to generate the virus AdmTyr-beta gal. This virus grows to high titer and maintains transcriptional specificity for pigment cell lineages. Strikingly, AdmTyr-beta gal is extremely active in human melanoma cells, in some cases exceeding the transcriptional activity of a cytomegalovirus promoter-driven recombinant beta-gal virus. Tissue specificity of gene expression is maintained, with very low levels observed in tumors and primary human cells derived from other lineages. These data provide evidence that it is possible to target human melanoma cells with great efficiency and specificity using high-titer recombinant adenovirus vectors.