The effects of three new catechol O-methyltransferase (COMT) inhibitors (nitecapone, entacapone and Ro 41-0960) were assessed on brain neurochemistry and psychomotor tests in levodopa/carbidopa-treated rats and mice. In neurochemical studies in rats, neither nitecapone (3 mg/kg) nor Ro 41-0960 (3 mg/kg) enhanced the levodopa/carbidopa (15/30 mg/kg)-induced dopamine levels in the hypothalamus and striatum, whereas 3-O-methyldopa (3-OMD) levels were suppressed. Adding clorgyline (8 mg/kg) elevated dopamine in the hypothalamus and striatum. 3,4-Dihydroxyphenylacetic acid and homovanillic acid levels were suppressed in the striatum and, to a lesser extent, also in the hypothalamus. Adding nomifensine (10 mg/kg) reversed the action of Ro 41-0960 on the 3-OMD levels and raised the homovanillic acid levels in the striatum. All COMT inhibitors (30 mg/kg) slightly enhanced levodopa/carbidopa (15/30 mg/kg)-induced hypoactivity in rats. Ro 41-0960 also augmented the effects of clorgyline and nomifensine on the locomotor activity. Levodopa/carbidopa (10/10, 25/25, 50/50, 100/100 mg/kg) dose-dependently decreased rectal temperature, rotarod time and spontaneous motility in mice. Additional COMT inhibition (30 mg/kg) had only minor effects on behavior. Ro 41-0960 (30 mg/kg) potentiated several behavioral effects of clorgyline (4 mg/kg) in the levodopa/carbidopa (10/10 mg/kg)-treated mice.