The role of a putative cholinergic control of ascending midbrain dopamine neurons was studied with biochemical methods in the unanaesthetized male albino rat. Post-mortem catechols were measured with high performance liquid chromatography with electrochemical detection. The acetylcholinesterase inhibitor physostigmine (0.5 mg/kg s.c.) enhanced L-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in both the corpus striatum and limbic areas (nucleus accumbens) after inhibition of aromatic amino acid decarboxylase with NSD-1015, indicating an enhanced synthesis of dopamine in these brain regions. The effect of physostigmine was blocked both in the corpus striatum and in limbic areas by the centrally penetrating muscarinic antagonist scopolamine (1.0 mg/kg s.c.). In contrast, the nicotinic antagonist mecamylamine (1.0 mg/kg s.c.) significantly reduced the stimulatory effect of physostigmine in limbic areas, but not in the corpus striatum. The present results suggest that ascending dopamine neurons are influenced by cholinergic synaptic transmission being mediated mainly by muscarinic receptors as regards the nigrostriatal system, and by both nicotinic and muscarinic receptors as regards the mesolimbic system. The nicotinic influence appears to primarily control phasic activity of the dopamine neurons.