The NADPH-dependent succinic semialdehyde reductase is one of the key enzymes in the brain GABA shunt, and it catalyzes the formation of the neuromodulator $gamma$-hydroxybutyrate from succinic semi aldehyde. This enzyme was inactivated by diethylpyrocarbonate (DEP) with the second-order rate constant of $1.1{ imes}10^3;M^{-1}min^{-1}$ at pH 7.0, $25^{circ}C$, showing a concomitant increase in absorbance at 242 nm due to the formation of N-carbethoxyhistidyl derivatives. Complete inactivation of succinic semialdehyde reductase required the modification of five histidyl residues per molecule of enzyme. However, only one residue was calculated to be essential for enzyme activity by a statistical analysis of the residual enzyme activity. The inactivation of the enzyme by DEP was prevented by preincubation of the enzyme with the coenzyme NADPH but not with the substrate succinic semialdehyde. These results suggest that an essential histidyl residue involved in the catalytic activity is located at or near the coenzyme binding site of the brain succinic semialdehyde reductase.