We have investigated the effect of the endogenous genotoxin malondialdehyde (MDA) on cell cycle kinetics and the expression and biochemical activity of several cell cycle regulatory proteins. MDA treatment of two human cell lines (RKO and H1299) resulted in a 3- to 6-fold elevation in the levels of the major detectable MDA-DNA adduct, M1G-dR. The increase in M1G-dR was accompanied by irreversible cell cycle arrest, elevation in p53 and p21 protein levels, and inhibition of cyclin E- and cyclin B-associated kinase activities. The decrease in cyclin E- and cyclin B-dependent kinase activities was caused by increased p21 and decreased cdc2 levels, respectively. Comparable levels of p21 induction were observed in RKO (wild-type p53) and H1299 (p53-null) cells. Thus, MDA was able to engage cell cycle checkpoint function in human cell lines when used at concentrations that produce M1G-dR levels of the same magnitude found in human tissues.