Inhibition of catechol-O-methyltransferase (COMT; EC 2.1.1.6) is a new therapeutic strategy in the treatment of Parkinson's disease. However, nothing is known about the effects of COMT inhibition on levodopa (L-dopa)-induced toxicity in dopamine (DA) neurons. Therefore we evaluated the effects of the selective COMT inhibitors Ro 41-0960, OR-486, and tolcapone alone and in combination with L-dopa in primary mesencephalic cultures from rat. Neither COMT inhibitor affected the growth of tyrosine hydroxylase immunoreactive (THir) cells with concentrations up to 10 microM when studied alone. However, Ro 41-0960 reduced the L-dopa-induced THir cell loss after 24 h in a dose-dependent manner, shifting the TD(50) value from 21 microM in the absence to 71 microM in the presence of 1 microM Ro 41-0960 (P <.01) without affecting survival of non-DA neurons. OR-486 and the clinically used COMT inhibitor tolcapone showed similar effects. In contrast, toxicity induced by D-dopa was not altered by COMT inhibitors. Furthermore, the primary metabolite of L-dopa formed by COMT, 3-O-methyldopa, and the methyl group donor S-adenosyl-L-methionine used by COMT did not alter THir neuron survival and L-dopa-induced toxicity, respectively, with concentrations up to 100 microM. These data demonstrate that COMT inhibition attenuates L-dopa toxicity toward DA neurons in vitro, but probably not by preventing 3-O-methyldopa production or cellular S-adenosyl-L-methionine depletion.