An aggregation substance of gramicidin S synthetases was found and purified by DEAE-cellulose chromatography and CM-chromatography from cell debris of Bacillus brevis Nagano. It specifically aggregated and inactivated gramicidin S synthetases 1 (GS1) and 2 (GS2). On the basis of amino acid composition analysis, reversed-phase HPLC, FAB mass spectrometry, amino acid sequence analysis, and antibacterial activity, this substance (GrS-aggregation substance) was identified as gramicidin S. A gramicidin S derivative bearing a lysine residue in place of one ornithine residue was also detected as a minor component of GrS-aggregation substance. The extent of the aggregation was dependent on the concentration and relative amount of gramicidin S. The inhibition of the enzyme activities was irreversible and the inhibition was proportional to the amount of gramicidin S, like the aggregation of the enzymes. The degree of GS2 inhibition in the amino acid-dependent ATP-PPi exchange reaction varied with the amino acids of gramicidin S and increased in order of the amino acid sequence of gramicidin S. The degree of inhibition of the overall synthesis of gramicidin S was the same as that in the leucine-dependent exchange reaction.