A series of N,N-dialkylnitrosamines (alkyl means methyl, ethyl, n-propyl, n-butyl or tert-butyl group) mono-substituted at the alpha-carbon with an acetoxy group, were tested for their mutagenic action in Salmonella typhimurium TA1530 in the presence or absence of a rat-liver supernatant from 9000 X g. The presumed released of methyl, ethyl, n-butyl and n-propyl carbonium ions from the corresponding alpha-acetoxy derivatives, either by enzymic cleavage or by non-enzymic hydrolysis of the ester group, caused high mutagenicity in the bacteria. As has been demonstrated for certain alpha-acetoxy compounds, the mutagenicity of these compounds was inversely related to their half-lives in aqueous media. N-(Acetoxy)methyl-N-tert-butylnitrosamine and a beta-acetoxy derivative of N,N-diethylnitrosamine were not mutagenic either in the presence or in the absence of hydrolysing rat-liver enzymes. These results support the hypothesis that alpha-carbon hydroxylation is one mechanism involved in the metabolic activation of N,N-dialkylnitrosamines.