Chemical mutagenesis in the mouse is a powerful approach for phenotype-driven genetics, but questions remain about the efficiency with which new mutations ascertained by their phenotype can be localized and identified, and that knowledge applied to a specific biological problem. During a global screen for dominant phenotypes in about 30,000 animals, a novel class of pigmentation mutants were identified by dark skin (Dsk). We determined the genetic map location, homozygous phenotype, and histology of 10 new Dsk and 2 new dark coat (Dcc) mutations, and identified mutations in Agouti (Met1Leu, Dcc4), Sox18 (Leu220ter, Dcc1), Keratin 2e (Thr500Pro, Dsk2), and Egfr (Leu863Gln, Dsk5). Cutaneous effects of most Dsk mutations are limited to melanocytes, except for the Keratin 2e and Egfr mutations, in which hyperkeratosis and epidermal thickening precede epidermal melanocytosis by 3-6 wk. The Dsk2 mutation is likely to impair intermediate filament assembly, leading to cytolysis of suprabasal keratinocytes and secondary hyperkeratosis and melanocytosis. The Dsk5 mutation causes increased tyrosine kinase activity and a decrease in steady-state receptor levels in vivo. The Dsk mutations represent genes or map locations not implicated previously in pigmentation, and delineate a developmental pathway in which mutations can be classified on the basis of body region, microscopic site, and timing of pigment accumulation.