Methamphetamine (MAP) administered in doses of 0.5 to 5 mg/kg i.p. to rats anesthetized with pentobarbital produced a shortening of the duration of loss of righting reflex. This analeptic effect of MAP was blocked by atropine but not by atropine methylbromide, indicating the central cholinergic nature of the response. This effect was also blocked by the D1 and D2 dopamine antagonists SCH 23390 (0.2 mg/kg) and raclopride (2 mg/kg), respectively. Pentobarbital decreased sodium-dependent high-affinity choline uptake (HACU) in frontal cortex and hippocampus as measured in synaptosomes from treated rats. MAP given to pentobarbital-narcotized rats restored HACU activity to nonanesthetized levels, but this restorative effect of MAP was blocked by SCH 23390 or raclopride. These data suggest that in addition to a cholinergic mechanism, the analeptic effect of MAP involves the dopamine system. alpha-Methyl-p-tyrosine, but not reserpine, pretreatment completely blocked the MAP analeptic response. In reserpinzed rats, MAP produced a markedly enhanced analeptic response. Studies of the effects of repeated administration of MAP on its analeptic activity were also undertaken in view of the well known sensitization to the locomotor and stereotypic effects of the amphetamines that occur with repeated intermittent administration. Rats pretreated daily with MAP (5 mg/kg) for 5 or 12 days showed neither tolerance nor sensitization to the analeptic effect of subsequent MAP administrations. 3H-quinuclidinyl benzilate-binding studies also showed no changes in muscarinic binding characteristics of membranes prepared from cortex or hippocampus of rats pretreated chronically with MAP. These and our earlier studies suggest that the analeptic effect of MAP is mediated via a dopaminergic-cholinergic mechanism.