Abstract Most of the natural melanin pigments consist of not only indolic eumelanin but also sulfur-containing pheomelanin. Previous methods for spectrophotometric assay of melanins use solubilization in alkaline media; a major disadvantage of these procedures is that they do not distinguish between eumelanin and pheomelanin. A spectrophotometric method for assaying eumelanin in tissue samples is described. Sepia melanin serves as a standard. Hair and melanoma samples were hydrolyzed in hot hydriodic acid, and insoluble eumelanic pigments were solubilized in hot sodium hydroxide in the presence of hydrogen peroxide and analyzed for absorbance at 350 nm ( A 350). The detection limit of eumelanin was ca. 2 μg. Eumelanins prepared from dopa, 5,6-dihydroxyindole and its carboxy derivative gave similar A 350 values. Mixed-type melanins prepared from dopa and various ratios of cysteine gave A 350 values inversely proportional to their sulfur contents. Excellent correlations were observed between A 350 values and contents of pyrrole-2,3,5-tricarboxylic acid, an oxidation product specific for eumelanin, in hair samples from sheep and humans of various colors and in melanomas and eyes from mice. The present method provides a specific and direct measurement of eumelanin contents in tissue samples.