Large releases of 1-penten-3-ol (pentenol) and 1-penten-3-one (pentenone) were recently observed from a variety of leaves subjected to freeze-thaw damage in the presence of oxygen. In order to understand the biochemical origins of these volatiles, soybean leaf extracts were used to determine if the formation of pentenol and pentenone can be explained by known O(2)-dependent lipoxygenase (LOX) reactions. Enzymatic formation of these C5 volatiles was found to be dependent on alpha-linolenic acid or the 13(S)-hydroperoxide of alpha-linolenic acid [13(S)-HPOT] and blocked by LOX inhibitors. Five soybean leaf LOX isozyme genes (VLXA, VLXB, VLXC, VLXD, and VLXE) were then expressed in Escherichia coli and used in in vitro incubations with 13(S)-HPOT to test for volatile formation. Each of the LOX isozymes catalyzed the formation of low levels of pentenol, but not pentenone. It therefore seems likely that the C5,13-cleavage activity of LOX is the direct source of abundant pentenol and the indirect source of pentenone observed upon leaf wounding.