Abstract We have previously shown that ethanol and acetaldehyde (ACE) potentiate MPTP toxicity in mice, selectively enhancing dopamine (DA) depletion in the striatum and markedly increasing loss of DA neurons in the substantia nigra. Several months after these combined treatments there is no evidence of any recovery. In the present study, we measured the accumulation of the MPTP toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP +) in both striatum and whole brain, after MPTP alone or after combined treatments with ethanol or acetaldehyde, in order to determine whether this enhancement of toxicity is caused by changes in the MPTP metabolism. We also investigated whether acetaldehyde interfered with the conversion of MPTP to MPP + by glial cells in vitro and studied its effects on the MPP + uptake and spontaneous release from mesencephalic DA neurons or striatal astrocytes in primary cell cultures from E13 mouse embryos. The results from the in vivo experiments indicated that relatively low doses of ethanol or acetaldehyde potentiate directly MPP + toxicity, apparently without interfering with its pharmacokinetics. However when higher doses of these drugs were administered, they also decreased MPP + clearance from the striatum. ACE also increased initial MPTP accumulation in the whole brain but failed to enhance MPP + levels, thus indicating thet ACE effect is not related to MPTP metabolism. In vitro studies confirmed that ACE does not modify MPTP metabolism in striatal or mesencephalic astrocytes in culture. In mesencephalic neuronal cultures ACE does not change the levels of MPP + uptake (MPP + is accumulated in putative DA neurons in vitro with a mechanism similar to that of the DA high affinity uptake) nor its spontaneous release. These results indicate that the slower MPP + clearance from the stratum after ACE is not related to a direct effect of ACE on DA neurons or astrocytes.