Eye tissues of tan-hooded, pink-eyed rats were shown in a previous report to become pigmented in short-term tissue cultures, and the phenomenon has now been analyzed in more detail. The rats, inbred and named the RCS strain, are homozygous for the recessive gene pink-eyed dilution, p, or an allele with similar properties. Eye tissues of postnatal p p rats and p p, p 1 p, and p 1 p 1 mice are minimally pigmented in vivo, and made little pigment when cultured in synthetic media lacking tyrosine. Pigmentation was strong when tyrosine was present in the medium, and the pigment was identified as melanin on the basis of color, staining properties, intracellular location, appearance in electron micrographs, and restriction to the cells of the iris, ciliary body, pigment epithelium, and choroid that contain melanin in pigmented animals. Pigmentation was obtained in primary cultures on collagen-coated coverslips and in organ cultures, in room air and in an atmosphere of 95% O 2-5% CO 2. An inductive mechanism for the response is unlikely because tyrosine-mediated pigmentation was not prevented by puromycin (up to 150 μg/ml) or actinomycin D (up to 10 μg/ml) in the culture medium. Failure of melanin-synthesizing enzyme activity because of membrane disarray within mutant melanosomes was ruled out because disordered granules, like the regular ones, appeared pigmented in electron micrographs. Competitive inhibition by excess phenylalanine was ruled out by phenylalanine measurements in vivo and by varying phenylalanine: tyrosine ratios in culture. Tyrosinase contains copper, and a gene-controlled copper deficit might be corrected in vitro by copper in ordinary culture media; however, tyrosine-mediated pigmentation was undiminished by the use of copper-free media. The p genes reduce pigmentation despite the presence of an apparently appropriate melanin-synthesizing machinery, possibly by causing tyrosine to be shunted to other pathways; exogenous tyrosine in vitro restored the normal phenotype.