Previous reports have documented an attenuated p53 response to DNA damage in hepatocytes isolated from enzyme-altered foci (EAF). Here, we have studied this p53 response in vivo in rats with EAF. These animals received repeated doses of diethylnitrosamine (DEN) for 6 weeks and a challenging dose 24 h before death. Liver sections were then analysed using an immunohistological procedure for p53, or a double-staining procedure for p53 and glutathione-S-transferase pi (GST-P). In control rats or rats with EAF not given the challenging dose of DEN, there was no p53 staining. In control rats, only given the challenging dose of DEN, there was a centrilobular p53 nuclear staining that co-localized with TUNEL staining. In an experiment involving four rats with EAF 389 +/- 39 hepatocytes/mm2 of non-EAF tissue stained positively for p53, while the corresponding value for EAF tissue was 27.6 +/- 7.5. Thus, p53-positive cells were 14.6-fold more frequent in non-EAF than in EAF tissue. In many EAF no p53-positive cells were seen at all and 83% of the EAF demonstrated <20% of the number of p53-positive cells seen in non-EAF tissue. Very few EAF had as high a proportion of p53-positive cells as did the average non-EAF tissue. EAF >0.06 mm2 had significantly fewer p53-positive cells than smaller EAF. The ratio of p53 expression in non-EAF tissue and large EAF was 32.6. In a control experiment, four EAF-bearing rats were used as donors to prepare primary cultures of hepatocytes. After 24 h of exposure to DEN, many of the cultured cells became p53-positive. Among GST-P-negative hepatocytes, 12.8% were p53-positive, whereas only 0.25% of the GST-P-positive hepatocytes were p53-positive. Literature data suggest that the altered xenobiotic metabolism in EAF may give rise to a 3-4-fold difference in DNA damage between non-EAF and EAF tissues. It is concluded that GST-P-positive EAF hepatocytes have an attenuated p53 response to DNA damage. This attenuated response may facilitate clonal expansion of EAF under stress induced by DNA-damaging chemicals.