Extracellular single-unit recordings and iontophoresis were used to compare the effect of a single administration of pertussis toxin (PTX, 1 microg), into midbrain dopamine (DA) nuclei (A9 and A10 regions), on the muscarinic, NMDA and DA receptor responses of midbrain DA cells in the anesthetized rat. Iontophoretic applications of DA, or apomorphine (50 microg/kg, i.v.), markedly reduced the firing of DA cells in control rats. In PTX-treated animals, these inhibitory responses were totally abolished, indicating that, in both DA nuclei, the inhibitory DA receptors are coupled to Gi/o proteins. In parallel, there was a significant decrease in the number of active DA cells per track which returned to baseline 5 weeks after the treatment. Applications of the muscarinic agonist oxotremorine M (OXO M) or of NMDA produced a potent increase in the firing of DA cells in control rats. DA neurons treated with PTX were still responsive to OXO M, although their sensitivity to the agonist was significantly reduced by 40%. In contrast, NMDA-induced activation remained unchanged, indicating that PTX did not non-selectively dampen all excitatory responses. Applications of cell-permeable cAMP derivatives did not change the basal firing of DA neurons. On the other hand, the phospholipase C inhibitors neomycin and ET-18-OCH3 (200 microg, i.c.v.), reduced significantly the activation of DA cells induced by OXO M. These data suggest that muscarinic activation of DA cells involves an M1-like receptor, possibly coupled to Gq/11 proteins, but also the participation of a PTX substrate.