The mutagenicity of hexachloro-1,3-butadiene and its S-conjugates 1-(glutathion-S-yl)-1,2,3,4,4-pentachloro-1,3-butadiene (GTB), 1,4-(bis-glutathion-S-yl-1,2,3,4-tetrachloro-1,3-butadiene (BGTB) and 1,4-(bis-cystein-S-yl)-1,2,3,4-tetrachloro-1,3-butadiene (BCTB) was investigated in Salmonella typhimurium TA100 using a modified preincubation assay. GTB was a direct-acting mutagen; the mutagenic potency of GTB was markedly enhanced by rat kidney microsomes or mitochondria and less so by cytosol. The bis-conjugates BGTB and BCTB were not mutagenic in the strains TA100, TA2638 and TA98. Purified HCBD was not mutagenic either without exogenous metabolic activation or with rat liver microsomes fortified with NADPH. Preincubation with rat liver microsomes and glutathione resulted in an unequivocal mutagenic activity of HCBD which was increased by additional inclusion of rat kidney microsomes. The cysteine conjugate beta-lyase inhibitor aminooxyacetic acid decreased the mutagenicity of HCBD and its S-conjugates. These results provide strong evidence that formation of the corresponding monoglutathione S-conjugate from HCBD and subsequent cleavage of this conjugate by gamma-glutamyltranspeptidase and beta-lyase may be responsible for the nephrocarcinogenicity of the parent compound in vivo, whereas formation of the bis-glutathione S-conjugate probably plays no role in the organ specific effects of HCBD.