The rat lymphocyte hprt assay measures in vivo mutagenicity by quantifying the frequency of 6-thioguanine-resistant (TGr) spleen lymphocytes cultured in vitro. In this study we have examined the types of mutations induced in the hprt gene of TGr lymphocyte clones from female Fischer 344 rats exposed to 100 mg/kg N-ethyl-N-nitrosourea (ENU). Hprt exons 3 and 8 were amplified from DNA extracted from each of 249 clones, and the resulting products were screened for mutant:wild-type heteroduplex formation by denaturing gradient gel electrophoresis. The analysis revealed 59 clones with mutations in exon 3, and 20 clones with mutations in exon 8. DNA sequence analysis of the heteroduplexes identified 84 mutations: all of the mutations were base pair substitutions, and 88% were mutations of A:T base pairs. At least 82% were induced independently. These results suggest that the mutations found in TGr rat lymphocytes from ENU-treated rats were due mainly to ethylthymidine adducts. In addition, a comparison of these results with previously reported in vivo ENU mutational profiles indicates that the types of mutation detected by heteroduplex screening of rat hprt exons 3 and 8 are representative of mutation in the entire protein coding sequence.