SUMMARY/ABSTRACT Cytochrome P450 (P450) enzymes are involved in the oxidations of ~95% of chemicals, including many drugs, steroids, and chemical carcinogens. The practical application of P450 research to drug metabolism has been a notable highlight in the pharmaceutical industry in the past three decades. Many of the leaders in this field have trained in this laboratory. Variations in P450s are issues in drug-drug interactions, endocrine diseases, cancer, and other medical problems. Despite some success using directed evolution and protein engineering, there is still much room for improvement in the development of P450s as biocatalysts. In this proposal, I plan to address several important questions about human P450 enzymes, including both microsomal and mitochondrial P450s: Is the Compound I intermediate (FeO3+) the only oxidant involved in P450 reactions, including steroid C-C bond cleavages? What are the physiological roles of the remaining human P450 orphans (i.e., the P450s that do not presently have a clear function)? What is the physiological relevance of P450 27C1 in human skin—we know the reactions catalyzed by this enzyme but we do not know the function. What are the roles of other binding proteins (other than for retinoids) in P450 oxidations of lipophilic physiological compounds? What is the molecular basis of P450 17A1-cytochrome b5 interactions and is there a potential for developing drugs for prostate cancer? Techniques to be used include organic synthesis, spectroscopy, enzyme kinetics, mass spectrometry, proteomics, X-ray crystallography, and cell and molecular biology. Collectively the proposed experiments will provide useful information about the functions and mechanisms of several human P450s and have continued relevance in the fields of endocrinology and drug metabolism.