Although phenobarbital, oxazepam and Wyeth 14,643 are carcinogens that do not form DNA adducts, they induce mutations in the Big Blue® transgenic mouse model. The mutations produced by these compounds were predominantly G→T and G→C transversions that we suspect arose from oxidative damage to DNA. To test this, we employed the single cell electrophoresis (Comet) assay that detects alkali-labile lesions in cells sustaining DNA damage. Human myeloid leukemia K562 cells were treated with non-cytotoxic doses of the above compounds for 3 h, then placed on slides containing low melting point agarose. Cells were lysed, exposed to alkaline buffer, electrophoresed and analyzed by microscopy for the existence of DNA damage. Extensive DNA damage, most likely due to the existence of single- and double-strand breaks and apurinic/apyrimidinic (AP) sites, was observed in cells exposed to oxazepam (1 mM) and Wyeth 14,643 (0.5 mM). On the other hand, damage of this sort was not observed in cells exposed to phenobarbital (1 mM). However, the addition of S9 liver extracts to cells exposed in the presence of phenobarbital resulted in significant amounts of DNA damage. We conclude from these studies that two of the three compounds evaluated in this study mediate their mutagenic effects through oxidative stress, but that the mechanism of DNA damage caused by phenobarbital differs from that elicited by oxazepam and Wyeth 14,643.