We purified from human fetal livers a form of cytochrome P450, termed HLp2, related to adult human liver cytochrome P450HLp by monitoring the purification procedures by immunoblots developed with a monoclonal antibody to HLp. The preparation of HLp obtained was judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to have an apparent monomeric molecular weight slightly greater than that of HLp, 51,500 versus 51,000. Immunoblot and Ouchterlony double-diffusion analyses indicated that HLp and HLp2 are immunochemically related. However, ferrous-CO versus ferrous difference spectra yielded different Soret maxima for HLp and HLp2 (448.5 and 449.5 nm, respectively). In addition, structural comparisons between HLp and HLp2 indicated that they were distinct isozymes. Specifically, the 28 amino acid amino-terminal sequence determined for HLp2 was found to be 79% homologous to that of HLp. In addition, peptide mapping of HLp and HLp2 by limited proteolysis with three proteases yielded similar but different patterns indicating that HLp and HLp2 are structurally distinct. These results demonstrate that HLp2 and HLp are highly related but distinct proteins and that HLp2 is a member of the steroid-inducible P450III gene family.