N-bromosuccinimide (NBS) inactivated completely malonyl-CoA synthetase from Rhizobium trifolii with a concomitant decrease in absorbance at 280 nm. The second-order rate constant for the inactivation was $1.8{ imes}10^{5}M^{-1}{cdot}min^{-1}$ at pH 6.9 and $30^{circ}C$. It was calculated from the spectral change at 280 nm that one tryptophan residue per molecule of the enzyme was modified. The intrinsic fluorescence study resulted that the modification of the enzyme did not cause any extensive conformational changes. The substrate, coenzyme A (CoA), afforded the protection against the inactivation of the enzyme caused by NBS. These results suggest that a catalytically essential tryptophan residue is located at the CoA-binding region of malonyl-CoA synthetase.