Ethylene oxide (EtO)-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were characterized in 28 independently derived 6-thioguanine-resistant human diploid fibroblast clones using polymerase chain reaction-based techniques and Southern blot analysis. Sequence analysis revealed one single base pair deletion and 13 base substitutions, nine of which were transversions: five AT-->TA, three GC-->TA and one GC-->CG. Four mutants were found to have GC-->AT transitions. Seven of the point mutations caused splicing errors. Six occurred in splice site sequences and one created a new splice acceptor site 16 bp upstream of exon 9. Three splice mutations were localized at the same site in the splice donor sequence of intron 8. Fourteen mutants had large HPRT gene deletions. In seven mutants the entire HPRT gene was deleted. The remaining deletion mutants had a truncated HPRT gene, where one or several exons were lost. These results show that EtO induces many different kinds of HPRT mutations, among which as many as 50% are large deletions.