Thymol and carvacrol are highly bioactive compounds often found in the essential oil of the culinary herbs, oregano (Origanum vulgare L.) and thyme (Thymus vulgaris L.). These two phenolic monoterpenes have a broad range of biological activities acting as antimicrobial compounds, insecticides, anti-oxidants and pharmaceutical agents. Previous studies of terpene biosynthesis in oregano, thyme and marjoram cultivars demonstrated that thymol and carvacrol are derived from γ-terpinene, which in turn is formed by a multi-product monoterpene synthase. However, the oxidation steps have not been previously described, aside from an older proposal that γ-terpinene is oxidized to thymol via the aromatic, cyclohexanoid monoterpene, p-cymene. As an approach to investigting the oxidation steps in thymol and carvacrol formation, eleven cytochrome P450 genes were isolated from oregano, thyme and marjoram (Origanum majorana) which could be assigned to five P450s, CYP71D178 through CYP71D182. The amino acid sequences of these P450s share high sequence identities with two limonene hydroxylases from mint. In addition, the transcript levels of most of the P450 genes are well-correlated with the occurrence of either thymol or carvacrol in various plant lines. Heterologous expression of CYP71D178-182 in yeast resulted in active enzymes which produced small amounts of thymol and carvacrol, but large amounts of p-cymene. However, p-cymene was not converted farther to thymol and carvacrol, suggesting its formation may be an artifact. To better test the role of these enzymes in planta, CYP71D178 and CYP71D180 genes were introduced into Arabidopsis. Feeding of γ-terpenene to transgenic Arabidopsis plants overexpressing these P450s resulted in the formation of thymol and carvacrol in good yield. These results indicate that thymol and carvacrol biosynthesis from γ-terpinene is catalyzed by cytochrome P450s in a two-step oxidation which might involve an allylic alcohol intermediate.