The mutagenicity of crotonaldehyde in Salmonella typhimurium TA100 cannot be demonstrated in the standard plate incorporation assay. However, as reported earlier by our group, this alpha, beta-unsaturated aldehyde is clearly mutagenic in the liquid assay modification of this testing procedure. Carried out because of the doubts recently expressed by Cooper et al. (Environ Mutagen 9:289-295, 1987) concerning the observed mutagenicity of crotonaldehyde in S. typhimurium TA100, this study confirms that this compound is clearly a direct (without activation by mammalian microsomes)-acting mutagen in S. typhimurium TA100 under appropriate conditions in the preincubation assay. The observed mutagenicity is increased by extended preincubation time and increased bacterial cell densities.