Mutagenicity and cytotoxicity of a series of N-alkyl-N-(alpha-hydroperoxyalkyl)-nitrosamines (alkyl = methyl, ethyl, propyl, butyl and tert-butyl) were tested in V79 Chinese hamster cells to examine the effects of alkyl chain length and the mode of substitution at the alpha-carbon bearing the hydroperoxy group on the biological activities. Among a series of hydroperoxymethyl compounds whose carbon bearing the hydroperoxy group is primary, the N-methyl compound was the most mutagenic and cytotoxic, and the biological activities of the compounds decreased in the following order: methyl much greater than ethyl greater than propyl greater than or equal to butyl greater than tert-butyl, the last one being nonmutagenic at the concentration tested. In a series of alpha-hydroperoxyalkyl compounds whose alpha-carbon bearing the hydroperoxy group is secondary, the N-methyl compound was also found to be the most mutagenic and cytotoxic, and the biological activities of the compounds decreased in the following order: methyl greater than ethyl much greater than propyl greater than butyl. A comparison of the biological activities of the corresponding compounds having the hydroperoxy group at primary or secondary alpha-carbon showed that the latter compounds were far more active than the former. A plot of the alkylating activity of the compounds toward 4-(rho-nitrobenzyl) pyridine after deoxygenation with NaHSO3 versus their mutagenic potency in V79 cells was linear, indicating that the chemical reactivity of the compounds plays an important role in inducing mutation in V79 cells.