Incubation of an NADPH-dependent succinic semi-aldehyde reductase from bovine brain with o-phthalaldehyde resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order kinetics with the second-order rate constant of 28 M(-1) s(-1). The inactivation was prevented by preincubation of the enzymes with NADPH, but not by succinic semialdehyde. There was a linear relationship between isoindole formation and the loss of enzyme activity. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the formation of one isoindole derivative per molecule of enzyme, which was formed from the reaction of cysteine and lysine residues with o-phthalaldehyde at or near the enzyme active site.