Hormonal steroids are biosynthesized from cholesterol by a number of conversions, which are catalyzed by enzymes belonging to two enzyme families, the steroid hydroxylases being cytochromes P450 (CYPs) and the hydroxysteroid dehydrogenases (HSDs) belonging to their own separate family. This article discusses the roles of the various hydroxylases, indicating their roles in the biosynthesis and breakdown of biologically active steroids, the regulation of their expression, their distribution over the various organ systems, and the effects of mutations in the genes encoding these proteins.