The enzyme polyphenol oxidase (PPO) is nearly ubiquitous in Kingdom Plantae and catalyzes the oxidation of phenolic compounds into highly reactive quinones. Although the functional importance of PPO in plants remains uncertain, a putative antipathogen role for walnut (Juglans regia) PPO was posited as early as 1911. However, despite the rich diversity of phenolics present in walnut leaves and hulls, walnut PPO has been little studied since the early 1900s. We cloned a PPO-encoding gene from a walnut pistillate flower cDNA library and designated the gene jrPPO1. Genomic Southern analysis demonstrated that jrPPO1 is the sole PPO gene in walnut. Transgenic tobacco (Nicotiana tabacum) plants expressing jrPPO1 display greater than 10-fold increases in leaf PPO activity compared with wild-type tobacco, demonstrating that jrPPO1 encodes a functional enzyme. The jrPPO1 protein is expressed primarily in the leaves, hulls, and flowers of walnut trees and is not regulated by wounding or methyl jasmonate. To examine whether walnut PPO could affect pathogen resistance, tobacco plants expressing jrPPO1 were challenged with Pseudomonas syringae pv. tabaci. Based on both symptom development and quantitative analyses of bacterial growth in planta, the PPO-expressing plants did not display increased resistance to this pathogen. Leaf extract browning assays indicated that tobacco leaves lack the endogenous phenolic substrates required for significant jrPPO1 activity and quinone production in planta.