We have developed a recombinant DNA shuttle vector that permits the molecular analysis of mutations induced in human cells by chemical or physical mutagens. The vector is able to replicate as a plasmid in Escherichia coli and in Epstein-Barr virus (EBV)-transformed human lymphoblastoid cell lines and contains the herpes simplex virus type 1 thymidine kinase gene (HSV tk) as the target for mutagenesis studies. After introduction of the vector into an EBV-transformed lymphoblastoid cell line (LCL-721) by electroporation, approximately equal to 2% of the transfected cells expressed the vector-encoded gene for hygromycin resistance. Plasmid DNA isolated from cells immediately after selection for hygromycin resistance (10 population doublings posttransfection) contained mutations in the HSV tk gene at a frequency of 6 X 10(-5). Treatment of plasmid-bearing LCL-721 cells with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of up to 15-fold in the frequency of mutations in the HSV tk gene. The dose-response for the induction of mutations in the plasmid-encoded gene closely paralleled that for the induction of mutations in the cellular gene for hypoxanthine (guanine) phosphoribosyltransferase.