The mutagenicity of the rat carcinogen acrylonitrile (ACN) and its metabolite 2-cyanoethylene oxide (CNEtO) was assessed in vitro in human lymphoblasts using the heterozygous thymidine kinase (tk) locus as a genetic marker. ACN was tested in both the presence and absence of an Aroclor-induced rat-liver homogenate S9. In the absence of S9, ACN was not mutagenic over the concentration range tested (0.4-1.5 mM X 2 h). In the presence of S9, the mutagenic response of ACN was enhanced, resulting in a significant response at a concentration of 1.4 mM X 2 h. CNEtO, the proposed ultimate mutagenic metabolite of ACN, induced a significant mutagenic response without activation at 100 microM and 150 microM X 2 h. Two phenotypic classes of spontaneous and CNEtO-induced tk-/- mutants were observed; one class of mutants (tkn) had a normal growth rate relative to wild-type while the second class (tks) grew at a slower rate. The molecular nature of these two phenotypic classes was investigated by Southern blot analysis. CNEtO-induced tkn mutant clones (11/12) and 7/9 tkn spontaneous mutants had no detectable alterations in their tk restriction fragment pattern. In contrast, 25/26 tks mutants analyzed (spontaneous and CNEtO-induced) had lost a 14.8-kb polymorphic fragment of the +tk allele.