Gramicidin S synthetase 1 (GS 1) [EC 5.1.1.11] (phenylalanine racemase) of Bacillus brevis contained about six sulfhydryl groups as determined by titration of the enzyme with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). Two types of sulfhydryl groups could be detected in the reaction with DTNB. One sulfhydryl group reacted rapidly with DTNB whereas the other five reacted more slowly with it. Phenylalanine racemizing activity was abolished on the rapid sulfhydryl modification with DTNB. When GS 1 of the wild strain was preincubated with phenylalanine at 37 degrees C in the presence of ATP, MgCl(2), and dithiothreitol (DTT), the rapid sulfhydryl modification with DTNB was prevented. When GS 1 was incubated with L-[14C]phenylalanine in the presence of ATP, MgCl(2), and DTT, 1 mol of L-[14C]phenylalanine was incorporated per mol of enzyme protein as an acid-stable phenylalanine thioester-enzyme complex. On the other hand, for GS 1 of a gramicidin S non-producing and phenylalanine racemization-lacking mutant of B. brevis, the substate protection against the rapid sulfhydryl modification was not detected and L-[14C]phenylalanine was not incorporated into the enzyme protein as the thioester complex. These results strongly suggest that one sulfhydryl group of GS 1 which reacts rapidly with DTNB is essential for the racemizing activity.