In the present study, retroviral vectors were used to stably transfer and express the cDNA encoding rabbit CYP4B1 in mouse C3H/10T1/2 cells. The replication defective retroviral vector was packaged in the ecotropic packaging cell line, GP+E-86, with infectious titer of approximately 1 x 10(6) cfu/mL. Infection, followed by selection with G418, showed an infection efficiency of approximately 70% for the recipient C3H/10T1/2 cells. Analysis of ten G418 resistant clones showed that the number of vector inserts ranged from 4 to 13 copies per cell genome. Each clone was positive for microsomal CYP4B1 protein as determined by immunoblotting. Cytochrome P450 4B1 activity was assessed by the cytotoxicity of 4-ipomeanol, a known substrate for P450 4B1 and a model compound for chemical-induced injury to the lung. The initial clonigenic assays showed that 100% toxicity occurred in all the clones after a 96-hr exposure to 250 microM 4-ipomeanol. Parental C3H/10T1/2 cells were resistant to 4-ipomeanol at concentrations as high as 1 mM. Two clones, designated No. 2 and No. 19, differing in levels of P450 4B1 protein, were characterized further for 4-ipomeanol and other chemical toxicities. A concentration-response study indicated 50% cytotoxicity at 4-ipomeanol concentrations of 1.5 micrograms/mL for clone No. 2 and 2.5 micrograms/mL for clone No. 19. A panel of agents representing the aromatic amines, some of which are known or suspected P450 4B1 substrates, were tested for cytotoxicity in clone No. 2. These agents included 2-aminoanthracene, 2-aminonaphthalene, 2-aminofluorene, 2-acetylaminofluorene and 4-aminobiphenyl. Only 2-aminoanthracene gave a clear cytotoxic response reducing the survival fraction of clone No. 2 to 50% at 0.2 micrograms/mL while affecting parental cells minimally. In vitro expression of CYP4B1 provides a new experimental system for further elucidating the cytotoxic and mutagenic effects of P450 4B1 substrates.