The metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) to reactive intermediates which covalently bind to cellular proteins was investigated. Isolated hepatocytes were used to determine whether protein alkylation is random in nature or whether it results in the alkylation of specific proteins. Isolated hepatocytes from rats treated with either ethanol (EtOH) or phenobarbital were incubated with the 14C-labeled nitrosamines for 1-3 h, after which the cells were separated from the incubation medium in order to distinguish secreted proteins from intracellular proteins. SDS-PAGE of the proteins in the medium followed by fluorographic analysis of the gels revealed that a heavily labeled protein was secreted into the medium which represents the predominantly labeled protein. Intracellularly, the major portion of the covalently bound label was found in the region of the gel where the cytochromes P-450 migrate. Pretreatment of the hepatocytes with diethyl maleate and buthionine sulfoximine to decrease the intracellular levels of glutathione had no effect on the labeling, indicating that glutathione does not protect cellular proteins from labeling by these carcinogens. Pretreatment of the cells with D-(+)-galactosamine to inhibit UDP-glucuronyltransferases resulted in a significant decrease in protein labeling by NDEA, suggesting that a glucuronide intermediate may be involved in the activation of NDEA to an alkylating species. The heavily labeled protein secreted into the incubation medium was identified as albumin on the basis of its apparent molecular weight of 66K, as determined by SDS-PAGE, and its cross-reactivity with anti-rat albumin IgG.