The enzymatic modification of tryptophan residues in Trp-Leu, Leu-Trp, Leu-Trp-Leu, and yeast mating hormone (alpha-factor), a tridecapeptide containing Trp-1 and Trp-3, has been investigated with tryptophan side chain oxidase I crystallized previously from Pseudomonas and tryptophan side chain oxidase II recently isolated from the same organism. With Trp-Leu as substrate, threo- and erythro-beta-hydroxytryptophan residues were identified as primary products and the former perferentially underwent second step dehydrogenation to give beta-ketotryptophan. Free threo- and erythro-beta-hydroxytryptophan, hitherto undescribed tryptophan derivatives, were isolated after enzymatic hydrolysis, identified, and characterized. Leu-alpha, beta-dehydrotryptamine, a new biochemical entity, was a sole main product of Leu-Trp. With Leu-Trp-Leu, diastereoisomeric Leu-beta-hydroxyl-Trp-Leu and Leu-alpha, beta-dehydro-Trp-Leu were formed simultaneously, the pH and ionic strength being determinants of the ratio of these products. Leu-threo-beta-hydroxy-Trp-Leu was also converted to Leu-beta-keto-Trp-Leu, but, distinct from NH2-terminal beta-hydroxytryptophan, both diastereoisomers underwent acid-catalyzed dehydration to give Leu-alpha, beta-dehydro-Trp-Leu. Thus, the mode of modification was shown to be prescribed by the site of tryptophan in peptides and the results were the same with either tryptophan side chain oxidase I or II, except that the latter was almost inactive on Trp-Leu. With any substrate, 3-indolecarboxaldehyde was a sole detectable by-product.