Acetylcarnitine is an endogenous substance, active in the CNS, but its exact role in neurotransmission is still unknown. The present study was made to analyze by means of iontophoretic administration on single neurones of the medullary-pontine reticular formation in the rat the effects of the D- and L-acetylcarnitine on spontaneous and glutamate-evoked firing and on responses to applications of some other putative transmitters, such as acetylcholine, 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) and noradrenaline. L-Acetylcarnitine increased both spontaneous and evoked discharges, and potentiated cholinergic and serotonergic responses. The D-isomer was found to be almost ineffective. Neuronal responses to L-acetylcarnitine were reduced by dihydro-beta-erythroidine and enhanced by eserine, while atropine was ineffective. L-Acetylcarnitine increased both excitatory and inhibitory responses to 5-HT and its effect on the increase in firing rate induced by serotonergic drugs was reduced by dihydro-beta-erythroidine, but not by atropine. L- and D-Acetylcarnitine did not affect GABAergic or noradrenergic transmission. These results suggest a stereospecific facilitatory action of acetylcarnitine on neuronal responses to acetylcholine and 5-HT. It is hypothesized that this action could be exerted at a presynaptic level, at least with regard to serotonergic-induced responses, and that nicotinic receptors may be involved in the synaptic mechanism of release or reuptake of both acetylcholine and 5-HT.