Adult human liver contains a form of cytochrome P450, termed HLp, that resembles the glucocorticoid-inducible cytochrome P450p in rat liver in its structure, function, and regulation and catalyzes the oxidation of such clinically important substrates as cyclosporin, nifedipine, erythromycin, and midazolam. Recent evidence, however, suggests that HLp may represent two or more closely related forms of cytochromes P450, one of which is termed P450nf. To search for additional members of the Class III human subfamily of HLp related genes, we screened a human liver cDNA library cloned in phage vector lambda gt11 with oligonucleotides and with a cDNA fragment related to HLp. We isolated a full-length cDNA (1709 nucleotides) encoding a new form of human cytochrome P450 termed HLp2. Analysis of HLp2 cDNA predicted a protein of 502 amino acids, weighing 57,294 Da 83% similar to HLp. HLp2 appears to represent a distinct gene as judged by partial sequence analysis of a cloned human gene and by hybridizations of Southern blots, under conditions of varying stringency, with a 3'-portion of HLp cDNA and with an oligonucleotide specific for HLp2. Northern blot analysis revealed that HLp/P450nf was present in all samples of liver mRNA from adult patients not treated with inducers of HLp, whereas HLp2 mRNA was undetectable in more than two-thirds. Human fetal liver RNA contained mRNA species 2.1 and 1.9 kb which hybridized with an HLp2 oligonucleotide. We conclude that HLp2 represents a third member of the Class III glucocorticoid-responsive gene family that is expressed in both fetal and adult human liver and may account for polymorphism in metabolism of clinically important drugs.