Combined cultures of human hepatocytes and human fibroblasts constitute a system composed entirely of normal human cells that can be used to investigate the mutagenicity of chemicals requiring metabolic activation. Addition of diethylnitrosamine (DEN) to this system resulted in mutations at the hypoxanthine-guanine phosphoriboribosyltransferase locus of the human fibroblasts. In separate experiments with cultures of hepatocytes alone, DEN induced unscheduled DNA synthesis (UDS) in the human hepatocytes. A comparative analysis of UDS and hepatocyte-mediated mutagensis indicates a great degree of similarity between the human and previously studied rat hepatocytes in their response to DEN in vitro.