We have measured the forward mutation rate at the hypoxanthine phosphoribosyltransferase (HPRT) gene of the human promyelocytic leukemia cell line HL-60 and have determined the molecular spectrum of spontaneous HPRT mutations in 45 independent 6-thioguanine-resistant HL-60 sublines. Four fluctuation tests using a total of 132 replicate HL-60 cultures revealed a mean forward mutation rate of HL-60 cells to thioguanine resistance of 1.7-6 x 10(-7)/cell/generation. Blot hybridization analysis of the X-linked HPRT gene using a human HPRT complementary DNA probe revealed abnormalities in HPRT gene structure and/or HPRT mRNA expression in 24 of 45 (53%) independent thioguanine-resistant HL-60 sublines. Six different classes of mutation were identified. The most prevalent (47%; 21 of 45 mutations) consists of mutations that are not detected by blot hybridization analyses and that do not disrupt HPRT mRNA production. These results suggest that a comparatively low forward mutation rate may be found in malignant human cells that exhibit both karyotypic and molecular evidence of genomic instability and that several different molecular classes of mutation may contribute to thioguanine resistance in HL-60, and perhaps in other, malignant human cells. The forward mutation assay system we have developed using the X-linked HPRT gene of HL-60 cells may be useful for analyses of the mutagenic potential and molecular spectrum of mutations produced by chemotherapeutic agents, suspected human mutagens and carcinogens, and phagocyte respiratory burst oxidants in human cells.