The tyrosinase gene encodes the key enzyme of melanin production and is tightly regulated during development. A yeast artificial chromosome covering the mouse tyrosinase gene has been shown to rescue completely the albino phenotype of recipient mouse strains, conferring copy number-dependent, position-independent expression. To investigate the presence of cis-acting regulatory elements responsible for the appropriate expression of the tyrosinase gene, DNase I hypersensitive site mapping was performed. A melanoma cell-specific DNase I hypersensitive site was identified at -12 kb upstream of the tyrosinase gene. Functional analysis of the corresponding cis-acting element in transgenic mice and transient transfection assays revealed properties of a strong cell-specific enhancer. RNA expression levels of the transgene correlate with copy number, which is reflected in coat colour and eye pigmentation of transgenic mice. Full enhancer activity in transient transfections is obtained with a minimal sequence of 200 bp. Protein binding analysis reveals the presence of a melanoma cell-specific complex which might contribute to the faithful expression of the tyrosinase gene.