Benzidine, a potent hepatocarcinogen in rodents, is readily metabolised to acetylated derivatives. In this study, the covalent binding of [3H-acetyl]N-acetylbenzidine and [3H-acetyl]N,N'-diacetylbenzidine to liver DNA in rats and hamsters was investigated. Binding to liver DNA of rats at 1 or 7 days after i.p. injection of N-acetylbenzidine was 2-fold higher than that observed in the liver DNA of hamsters which had been similarly treated. Analysis of enzymically hydrolysed DNA from both species indicated the presence of a single adduct which co-eluted with N-(deoxyguanosin-8-yl)-N'-acetylbenzidine. In vitro treatment of rat or hamster liver slices with N-acetylbenzidine also resulted in covalent binding to hepatic DNA and the identical DNA adduct was detected at levels comparable to that observed in vivo. When N,N'-diacetylbenzidine was injected i.p. into rats, a comparatively low level of binding to liver DNA was observed. Following enzymic hydrolysis, the major DNA adduct detected by h.p.l.c. analysis was again N-(deoxyguanosin-8-yl)-N'-acetylbenzidine accompanied by a small amount of N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine. In vitro incubation of N,N'-diacetylbenzidine with rat liver slices resulted in DNA binding levels similar to that observed with N-acetylbenzidine. In contrast to what was found in vivo, N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was the major adduct detected in DNA from rat liver slices. These data suggest that both N-hydroxy-N'-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidine are proximate carcinogenic species of benzidine, with N-hydroxy-N'-acetylbenzidine the more important. The low level of N-(deoxyguanosin-8-yl)N,N'-diacetylbenzidine observed in vivo may be due to its rapid repair. Alternatively, N-sulphonyloxy-N,N'-diacetylbenzidine, which would produce this adduct on reaction with DNA, may be efficiently detoxified in vivo.