T-cell activation by malignant melanoma would be anticipated to stimulate T-cell proliferation, which in turn has been associated with increasing the likelihood of somatic gene mutation. The purpose of this study was to test the hypothesis that in vivo hypoxanthine guanine phosphoribosyltransferase (hprt) mutant frequencies (MFs) are increased in peripheral blood T-cells from melanoma patients compared to normal controls. Assays were made of 48 peripheral blood samples from melanoma patients with stage 3 (13 patients) and stage 4 (35 patients) disease, 38 normal controls, and of nine tumor bearing lymph nodes. The mean hprt log(10)(MF) in patient peripheral blood was -4.77 (geometric mean hprt MF=17.0x10(-6)) compared to a mean hprt log(10)(MF) of -4.87 (geometric mean hprt MF=13.5x10(-6)) in controls. Although modest, this difference is statistically significant both by t-test (P=0.049) and after adjustment for covariates of age, gender, and cigarette smoking by regression analysis (P=0.001). Among the melanoma patients, the mean log(10)(MF) for the 17 patients who had received potentially genotoxic therapies was not significantly different from the mean log(10)(MF) for the 31 patients not receiving such therapies. The hprt MFs in the nine tumor bearing nodes were compared with MFs in peripheral blood from the same patients and revealed a non-significant (P=0.07) trend for increasing MFs in blood. Furthermore, analyses of T-cell receptor gene rearrangement patterns revealed hprt mutants originating from the same in vivo clone in both peripheral blood and a tumor-bearing node. The finding of elevated hprt MFs not entirely explained by genotoxic therapies in patients compared to controls can be explained either by hypermutability or in vivo T-cell activation. The similar MFs in peripheral blood and tumor bearing lymph nodes, as well as the finding of mutant representatives of the same in vivo T-cell clone in both locations, support monitoring peripheral blood to detect events in the nodes. If in vivo proliferation accounts for the current findings, the hprt deficient (hprt-) mutant fraction in blood may be enriched for T-cells that mediate the host immune response against malignant melanoma. Further studies will characterize the functional reactivity of hprt mutant isolates against melanoma-related antigens.