Abstract The neuroprotective effects of MK-801, a non-competitive antagonist of the N- methyl- d-aspartate (NMDA) receptor/channel, were assessed in a culture model which reproduces in vitro the selectìve degeneration of mesencephalic dopaminergic neurons seen in parkinsonian brains. Dissociated mesencephalic cells derived from rat embryonic brains were subjected for 24 h to intoxication by the 1-methyl-4-phenylpyridinium (MPP +), the active metabolite of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPP + at 3 and 10 μM produced selective and dose-dependent damages to dopaminergic neurons as quantified by the loss of the number of TH immunoreactive cells and the loss of [ 3H]DA uptake whereas other cell types remained unaffected. MK-801 at 3 and 10 μM failed to rescue degenerating dopaminergic neurons in presence of MPP +. At 50 μM, i.e. the highest concentration that is not toxic by itself in this culture system, MK-801 was also found ineffective. Furthermore, degree of dopaminergic cell damage was not reduced when repeated additions of the glutamate antagonist (10 μM/6 h for 24 h) were performed during exposure to MPP + or when mesencephalic cultures were left after intoxication for up to 2 days in a culture medium still supplemented with MK-801 but free of toxin. In accordance with these results, MK-801 did not affect significantly the uptake of [ 3H]DA in control cultures, thereby suggesting that this compound cannot prevent intracellular accumulation of MPP + within dopaminergic neurons. At higher concentrations of MPP + (100 μM) tested, toxic effects were seen toward dopaminergic neurons and non-dopaminergic cells as quantified by Trypan blue dye accumulation and loss of [ 3H]GABA uptake. Similarly, widespread degenerative changes were observed with high levels of glutamate (4 mM), the brain endogenous agonist of the NMDA receptor/channel. Yet, degree of cell injury was not reduced in presence of MK-801 in both situations. Meanwhile, to establish that MK-801 was pharmacologically active in our incubation conditions, we showed that the different batches of this compound which have been found ineffective in mesencephalic cultures were indeed capable of preventing neurotoxic effects of glutamate toward dissociated cortical cells. Altogether, these results indicate that MPP + detrimental effects in vitro are not related to excitotoxicity and/or that MK-801 does not interfere with MPP + toxic scenario. They do not support the idea that glutamate antagonists could be used to prevent dopaminergic cell degeneration in Parkinson's disease.