Malondialdehyde (MDA), a product of lipid peroxidation and prostaglandin biosynthesis, has been reported to be mutagenic and carcinogenic. Recent evidence suggests, however, that strongly mutagenic impurities are generated during the preparation of MDA that may contribute to the observed biological activity. Since MDA is widely produced in animal tissue it is important to establish whether it is actually mutagenic and carcinogenic. We have utilized three complementary methods for the preparation of highly purified MDA for biological testing. These are chromatographic purification of the sodium salt of MDA, sublimation of the free acid of MDA, and basic hydrolysis of beta-(p-nitrophenoxy)acrolein. The latter is a unique method that we have developed specifically to generate MDA under non-acidic conditions where it is stable. MDA prepared by each method induced approximately 5 revertants/mumol in Salmonella typhimurium his D 3052. This unequivocally demonstrates that MDA is a weak mutagen.