Previous studies have suggested that fish have oncogene sequences homologous to those found in mammalian and avian species. We were the first to confirm the presence of fish oncogenes by isolating and sequencing the c-myc gene from rainbow trout (Van Beneden et al., 1986). Other fish genes homologous to known mammalian oncogenes were identified by Southern blot hybridization. In order to examine the role of fish oncogenes, Southern blots were prepared using DNA digests from tumor and normal tissue. Hybridization to known oncogene sequences did not detect rearrangements or gene amplifications. In order to detect other oncogenes, we developed a transfection system in which fish DNA in the presence of calcium phosphate was transfected into NIH 3T3 cells. The transforming ability of fish tumor DNA was examined by standard focus assay, nude mouse assay, and colony selection assay. DNA from diethylnitrosamine-induced mesothelioma in medaka was the most efficient in transformation of NIH 3T3 cells. Secondary transfectants caused formation of tumors in nude mice of greater than 20mm in 1-1/2 weeks following injection. Southern blot analysis of these transfectant DNAs hybridized to medaka genomic DNA probe showed bands present in tumor-induced transfectants. No bands were present in DNA from NIH 3T3 controls and cells transfected with non-tumorigenic medaka DNA. This suggests the presence of specific fish sequences in transformants. These sequences do not appear to be homologous to K-ras, H-ras, N-ras, c-myc m-met or v-erbB. This study will continue to identify, clone, and sequence the transforming gene from fish in order to examine its role in tumor formation.