A cDNA coding for a P450, designated IIF1, was isolated from a human lung lambda gt11 library by screening with a human IIC9 cDNA probe. The cDNA-encoded IIF1 protein had 491 amino acids and a calculated molecular weight of 55,507. IIF1 cDNA, expressed by using a vaccinia virus vector, produced a cytochrome with a lambda max of 454 nm when reduced and complexed with carbon monoxide. This enzyme was able to dealkylate ethoxycoumarin, propoxycoumarin, and pentoxyresorufin but possessed no activity toward ethoxyresorufin and only trace dearylation activity toward benzyloxyresorufin. A variant cDNA, designated IIF1v, was isolated that was identical with IIF1 except for the loss of two segments of 161 and 388 bp within the cDNA coding region. Two mRNAs, consistent with the predicted size of the IIF1 and IIF1v transcripts, were found at very low abundance in lung specimens by Northern blot analysis. A 2-kb transcript, hybridizing with the human IIF1, was also detected as an abundant mRNA in rat lung. The CYP2F gene subfamily was localized to human chromosome 19 and mouse chromosome 7. On the basis of Southern blotting analysis with multiple restriction enzymes, we conclude that the CYP2F1 gene is flanked by a second highly similar gene.