DNA damage induced by tobacco-related nitrosamines was quantitatively determined in animal-mediated DNA-repair assays with Escherichia coli K-12 strains. Intraperitoneal administration of N-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosopyrrolidine (NPYR) caused dose-dependent genotoxic effects in indicator bacteria recovered from various organs of nitrosamine-pretreated mice. Oral administration of ethanol (6.3 g/kg body wt) 1 h prior to administration of NNN, NNK or NPYR resulted in a substantial reduction of genotoxicity that was more pronounced in the liver as compared to lungs, spleen, kidneys and blood. However, when the same ethanol dose was given 26 h before NNN or NPYR, an increase of DNA damage was found, that was, in most cases, higher in the kidneys than in the liver. Significant enhancement of genotoxic activity was also measured in lungs and spleen, whereas only a marginal increase was detectable in the blood. Repeated administration of smaller ethanol doses (2.0 g/kg body wt at 12 h intervals) for 4 days caused a comparable increase. Similar enhancement of genotoxicity was also measured when acetone (3.5 g/kg) was given orally 15 h before the nitrosamine administration. The stimulating effect of ethanol was dose dependent and was absent when the alcohol was administered 60 h prior to the nitrosamine. Neither ethanol nor acetone had an effect on the genotoxicity of NNK under identical experimental conditions. The same E. coli K-12 strains were used to test NNN, NNK and NPYR in in vitro assays. The ranking order of activation capacity was liver S-9 > kidney S-9 > lung S-9 for all three nitrosamines. Blood S-9 did not markedly activate the nitrosamines. S-9 mixtures prepared from mice that had been treated with ethanol (6.3 g/kg body wt) for 26 h before death activated NNN and NPYR more efficiently than those S-9s from untreated animals. The increase of genotoxic activity was more pronounced with S-9 from kidneys and lungs than from liver. No difference was seen with S-9 from ethanol-treated and untreated mice with NNK.