Mutation to ouabain resistance and cytotoxicity were tested in V79 Chinese hamster cells after the cells had been treated for 2.5 hr with a series of N,N-dialkylnitrosamines (alkyl=methyl, ethyl, propyl, butyl or tert-butyl) monosubstituted at the alpha-carbon with an acetoxy group. The effects of the length of alkyl chain and the mode of substitution with the acetoxy group on the cytotoxicity and mutagenicity were examined. In the series of N-alkyl-N-(acetoxy-methyl) nitrosamines with an acetoxy group at the primary alpha-carbon, the methylating compound N-methyl-N-(acetoxymethyl) nitrosamine was the most cytotoxic and mutagenic, and the biological activities decreased in the order of ethyl, butyl and propyl homologs. N-tert-Butyl-N-(acetoxymethyl) nitrosamine was not mutagenic at the concentration tested. At equitoxic concentrations, N-ethyl-N-(acetoxymethyl) nitrosamine was found to be the most mutagenic. Of the two N-alkyl-N-(alpha-acetoxybutyl) nitrosamines having an acetoxy group at the secondary alpha-carbon, N-methyl-N-(alpha-acetoxybutyl) nitrosamine was more cytotoxic and mutagenic than N-butyl-N-(alpha acetoxybutyl) nitrosamine. A comparison of the corresponding N-alkyl-N-(acetoxymethyl) nitrosamines and N-alkyl-N-(alpha-acetoxybutyl) nitrosamines showed that the latter had stronger activities. A plot of the mutation frequency versus the ability of the alpha-acetoxy compounds to alkylate 4-(p-nitrobenzyl) pyridine was linear. This indicates that the chemical reactivity of the compounds plays an important role in inducing mutation in V79 Chinese hamster cells.