The reaction product of N-methyl-N-nitrosourea (MNU)) with DNA, O6-methylguanine (O6-MeG), is responsible for the mutagenic and carcinogenic effects of this carcinogen. These involve activation of the H-ras proto-oncogene in rat mammary tumors by MNU, with a high frequency of GC to AT transitions in codon 12 of this gene. The present study aimed to investigate the types and position specificities of mutations induced by MNU in another gene, the hprt gene of V79 Chinese hamster cells. Furthermore, since processes involved in the expression of genetic damage, e.g. the state of the DNA precursor pool, have been suggested to be important factors in carcinogenesis, the mutagenic specificity of MNU was also studied in the presence of an imbalanced nucleotide pool. Isolation of Independent hprt mutant clones from three groups treated in different manners was performed. Two different doses of MNU and a low dose of MNU in combination with hydroxyurea (HU) were employed. Comparison of the results with the two doses of MNU did not indicate any shift in mutation specificity. The majority of the mutations induced by MNU were base substitutions, mostly transitions of GC to AT showing high affinity for the middle base in 5′-purine-G-N-3′ sequences (15/18) in the non-transcribing strand, suggesting a difference in repair capacity for the two strands. The relatively high frequency of the base substitutions resulting in splicing defects is explained by the presence of a consensus sequence (5′-purlne-g-N-3′) in the splice sites of the hprt gene. The results from the HU/MNU group showed a few more GC to TA transversions, though not statistically significant, which may be caused by a shift from miscoding to non-coding recognition of the O6MeG lesion. The same reactive decomposition products formed from MNU are also formed from a variety of other carcinogenic compounds, e.g. N-methyl-N'-nitro-N-nitrosoguanidine, dimethylnitrosamine, nitrosocimetidine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, suggesting that our findings concerning the mutagenic specificity of MNU in mammalian cells are valid also for these other compounds as well.