The enzymatic synthesis of l‐tryptophan and its derivative 5′‐hydroxy‐l‐tryptophan labeled with deuterium and tritium at the α‐carbon position is reported. The mixture containing S‐methyl‐l‐cysteine, indole or 5‐hydroxyindole dissolved in deuteriated or tritiated water has been converted to [2‐2H]‐l‐tryptophan, [2‐3H]‐l‐tryptophan, 5′‐hydroxy‐[2‐2H]‐l‐tryptophan, and 5′‐hydroxy‐[2‐3H]‐l‐tryptophan, respectively, in a one‐pot reaction using the enzyme tryptophanase. The same reaction carried out in heavy water with THO added yielded either doubly labeled [2‐2H/3H]‐l‐tryptophan or 5′‐hydroxy‐[2‐2H/3H]‐l‐tryptophan. Copyright © 2003 John Wiley & Sons, Ltd.