Maize (Zea mays) produces zealexins as phytoalexins, with the inducible production of these antibiotics providing biochemical protection against fungal infection. However, the biosynthesis of these sesquiterpenoids has remained unclear. In particular, it is unclear how the olefinic precursor, (S)-β-macrocarpene produced by the characterized maize sesquiterpene synthases TPS6/11, is further elaborated to form the bioactive zealexins. The first step is likely to be conversion of carbon-15 (C15) from a methyl group to a carboxylic acid by a cytochrome P450 mono-oxygenase (CYP). In this study, CYP71Z18, whose transcription is strongly induced by fungal infection, was found to catalyze oxidation of C15 in (S)-β-macrocarpene, forming zealexin A1. The inducible transcription of CYP71Z18 matches that observed for TPS6/11 and the accumulation of zealexins, which is consistent with a role for CYP71Z18 in sesquiterpenoid phytoalexin production. This completes identification of zealexin A1 biosynthesis, and represents the initial CYP identified for the production of maize terpenoid phytoalexins.