The formation and stability of DNA—protein crosslinks (DPXLs) formed by incubation of pUC13 plasmid DNA and calf thymus histones with 1–100 mM acetaldehyde was studied using a filter binding assay. DPXLs were formed at a rate of 127 DPXLs/plasmid molecule/mmol acetaldehyde in a reaction containing 1 μg of histones and 0.33 μg of DNA at 37°C for 1 h. Acetaldehyde-induced DPXLs were unstable at 37°C, with loss of up to 75% by 8 h. Crosslink formation was significantly higher at lower pH, with 3- and 2-fold higher levels at pH 5 and 6 respectively than at pH 7.5. Induction of DPXL formation by 1–100 mM vinyl acetate in the presence of rat liver microsomes was observed at 37°C over 3 h. DPXL accumulation followed S-phase enzymatic kinetics, with a rate of formation of 1.1 DPXLs/plasmid molecule/mmol vinyl acetate/μg microsomal protein/μg DNA. Vinyl acetate was unable to cause formation of DPXLs in the absence of microsomes. A carboxylesterase inhibitor, bis-(p-nitrophenyl) phosphate, was able to block DPXL formation by vinyl acetate and microsomes. This work supports the hypothesis that DPXL formation by vinyl acetate requires microsomal metabolism to acetaldehyde, which is the active crosslinking agent.