A growing body of evidence indicates that nitric oxide (NO) plays important signaling roles in plants. However, the enzyme(s) responsible for its synthesis after infection was unknown. Here, we demonstrate that the pathogen-induced, NO-synthesizing enzyme is a variant form of the P protein of glycine decarboxylase (GDC). Inhibitors of the P protein of GDC block its NO synthase (NOS)-like activity, and variant P produced in E. coli or insect cells displays NOS activity. The plant enzyme shares many biochemical and kinetic properties with animal NOSs. However, only a few of the critical motifs associated with NO production in animals can be recognized in the variant P sequence, suggesting that it uses very different chemistry for NO synthesis. Since nitrate reductase is likely responsible for NO production in uninfected or nonelicited plants, our results suggest that plants, like animals, use multiple enzymes for the synthesis of this critical hormone.