In an attempt to elucidate mechanisms underlying the variation in radiosensitivity during the cell cycle, mutations in the HPRT gene were selected with 6-thioguanine, quantified and characterized in synchronous human bladder carcinoma cells (EJ30-15) that were irradiated in G1 or S phase with 3 or 6 Gy. Synchronous cells were obtained by mitotic selection, with approximately 98% of the cells in G1 phase when they were irradiated after 3 h of incubation, and 75% in S phase when they were irradiated after 14 h of incubation. The mutant frequencies were approximately 4-fold higher (P < 0.01) when cells were irradiated in G1 phase compared with S phase, and the lowest frequency (1.5 x 10(-5) for 3 Gy during S phase) was approximately 10-fold higher than the spontaneous frequency. Exon analysis by multiplex polymerase chain reaction was performed on DNA isolated from each independent mutant. The different types of mutants were categorized as class 1, which consisted of base-pair changes or small deletions less than 20 bp; class 2, which consisted of deletions greater than 20 bp but with one or more HPRT exons present; and class 3, which consisted of deletions encompassing the entire HPRT gene and usually genomic markers located 350-750 kbp from the 5' end of the gene and/or 300-1400 kbp from the 3' end. A "hotspot" for class 2 deletions was observed between exons 6 and 9 (P < 0.01). For cells irradiated during G1 phase, the percentages for the different classes (total of 78 mutants) were similar for 3 and 6 Gy, with a selective induction of class 3 mutants (34-38%) compared with spontaneous mutants (3%, total 20). When S-phase cells were irradiated with 3 Gy, there were fewer class 1 mutants (21%, total 37) than when cells were irradiated in G1 phase with 3 Gy (45%, total 42) (P < 0.01). The greatest change was observed when the dose was increased in S phase from 3 Gy to 6 Gy (total of 43 mutants), with the frequency of class 2 mutants decreasing dramatically from 30% to 1% (P < 0.005). A similar decrease in class 2 mutants with an increase in dose has been observed by others in asynchronous cultures of normal human fibroblasts. We hypothesize that these differences occur because: (a) there is more error-free repair of double-strand breaks (DSBs) during S than G1 phase; (b) a single DSB within the HPRT gene causes a class 2 mutation or a certain percentage of class 1 mutations, while two DSBs, with one in each approximately 1-Mbp region 5' and 3' of the gene, cause a class 3 mutation; and (c) a repair process that is induced when the dose during S phase is increased from 3 to 6 Gy results in a preferential decrease in class 2 mutations.