The aim of this study was to determine whether o-chlorobenzylidene malononitrile (CS) exhibits any genotoxic activity towards Salmonella or mammalian DNA in vivo. CS was synthesized with a [14C]-label at the benzylic carbon atom. It was administered i.p. at a dose level of 13 mg/kg (1 mCi/kg) to young adult male rats. Liver and kidney DNA was isolated after 8,25, and 75 h. The radioactivity was at (liver, 8 and 75 h) or below (all other samples) the limit of detection of 3 dpm. Therefore, a possible binding of CS to DNA is at least 105 times lower than that of the strong hepatocarcinogen aflatoxin B1, and 4,000 times lower than that of vinyl chloride. In contrast to this lack of DNA binding, but in agreement with the chemical reactivity of CS, a binding to nuclear proteins could be detected with specific activities ranging between 50 and 121 dpm/mg for liver and between 3 and 41 dpm/mg for kidney. Protein binding could well be responsible for its pronounced cytotoxic effects. CS was also tested in the Ames Salmonella/microsome assay. Strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were used with or without pre-incubation. Only with strain TA 100 and only without pre-incubation, a doubling of the number of revertants was detectable at the highest dose levels used, 1,000 and 2,000 μg CS per plate. With pre-incubation of TA 100 with CS, a slight increase of the number of revertants was seen at 100 and 500 μg per plate, and a subsequent fall below control values at 1,000 μg. A check for the number of surviving bacteria revealed a strong bacteriotoxicity of the higher doses of CS so that the calculated mutation frequencies, i.e., the number of revertants per number of surviving bacteria, increased with doses up to 500 μg. This toxicity could be counteracted in part by the addition of increasing amounts of rat liver microsomes. In the view of these results, and taking into account the rare and low exposure of man, it is concluded that CS will not create a risk for the induction of point mutations or of carcinogenic processes mediated by DNA binding.