Xiphophorus interspecies hybrids provide genetically defined models of both spontaneous and inducible melanomagenesis. In both models, backcrossing F(1) hybrids of different strains of X. maculatus and X. helleri to a X. helleri parental fish results in segregation of melanoma susceptibility, fitting a Mendelian two-gene inheritance model. The sex-linked Xmrk oncogene is required for melanoma development in both crosses. The Xiphophorus CDKN2A/B gene, which is homologous to mammalian CDKN2A/B cyclin-dependent kinase inhibitors (p16 and p15), is a candidate melanoma susceptibility gene. In this model, tumor susceptibility segregates with homozgyosity for CDKN2A/B from the recurrent X. helleri parent in backcross hybrids. We found that both CDKN2A/B mRNA and protein are highly overexpressed in melanoma. Because the p13 protein product of CDKN2A/B is a putative regulator of the G1 checkpoint, we investigated expression of other components of Xiphophorus G1 checkpoint control. By real-time PCR analysis, retinoblastoma gene (RB) is consistently expressed twofold higher in both tumors and melanized skin than in normal tissue, indicating that RB is not downregulated by the overexpression of CDKN2A/B in Xiphophorus melanoma. We also found a significant correlation between the quantitative level of CDKN2A/B and Xmrk RNA in tumors, suggesting a functional relationship between Xmrk and CDKN2A/B expression. Although X. helleri CDKN2A/B protein contains a non-conservative substitution, the biochemical function appears to show little overt defect. These studies indicate that in Xiphophorus melanoma, CDKN2A/B is functionally insufficient to mediate cell-cycle arrest in the presence of Xmrk.