Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen in zebrafish (Danio rerio) was performed to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic eye assay that models this second hit. This assay is a visible test for loss of wild-type gene function at a single locus, golden, representative of genome- wide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for seven of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30-34 months was increased 10-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of mechanisms of somatic loss of gene function, and how those mechanisms contribute to cancer susceptibility.