This in vitro study using human liver enzymes was undertaken in order to compare the mechanism of metabolic reduction of timiperone, a potent butyrophenone neuroleptic, with that of haloperidol. Conversion of timiperone to reduced timiperone in liver cytosol was confirmed. The carbonyl reductase inhibitors (menadione IC50 5-18 μM; ethacrynic acid IC50 26-51 μM) potently inhibited both timiperone reductase and haloperidol reductase activity, while 4-methylpyrazole (alcohol dehydrogenase inhibitor) had no effect and phenobarbital (aldehyde reductase inhibitor) had a weak inhibitory effect. The formation of reduced timiperone was highly correlated with the formation of reduced haloperidol(r=0.87, n=6, P<0.02). Timiperone reductase activity was approximately 40% lower than haloperidol reductase activity (at a substrate concentration of 100 μM, two-tailed t-test, P<0.05). The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of reduced timiperone formation were much lower than reduced haloperidol formation (Km values: 29.7±15.1 versus 381.3±1.1 μM, n=3, P<0.01; Vmax: 0.81±0.19 versus 6.00±1.47 nmol/mg/min; n=3, P<0.05). However, the ratio Vmax/Km (clearance) for timiperone was 1.3-2.4 times higher than for haloperidol, indicating that metabolic clearance of timiperone by carbonyl reductase may be similar to or slightly higher than for haloperidol.