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 was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide 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 7 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 9.6-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 the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility.