We have analyzed mutations in exon 8 of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in T-lymphocytes from the spleens of ethylnitrosourea-treated female rats. Presumptive hprt- mutants were isolated by clonal growth in the presence of 6-thioguanine. DNA from 6-thioguanine-resistant colonies was amplified by the polymerase chain reaction using intronic primers flanking hprt exon 8. The identification of mutant sequences and the separation of mutant DNA from contaminating wild-type DNA was accomplished by denaturing gradient gel electrophoresis. Of 118 clones analyzed, 19 contained mutations and DNA sequence analysis identified eight unique sequence alterations. We also used single-strand conformation polymorphism analysis to screen for mutations in the same fragment of the hprt gene. This analysis was less successful than denaturing gradient gel electrophoresis in detecting the eight unique mutations. The procedures described here may represent a useful approach for studying the mechanisms of in vivo mutation.