The aim of the present study was to explore possible prejunctional effects mediated by impulse activity of sympathetic terminals on evoked acetylcholine release in an organ innervated by the autonomic ground plexus. Rabbit atria were isolated with the extrinsic right vagus and sympathetic nerves intact and perfused with Tyrode solution. Acetylcholine overflow was determined after labelling of the transmitter stores with [14C]choline and fractionation of the radioactivity on cation exchange columns. The overflow of endogenous noradrenaline was measured by HPLC and electrochemical detection. The vagus nerve was stimulated at 2 Hz for 3 min four times at intervals of 10 min. During the second stimulation the postganglionic sympathetic nerves were stimulated (2 Hz, 3 min) in such a way that the impulses preceded the vagus stimuli by a fixed time interval which was varied in different experiments (0, 7, 19, 50, 132, and 350 ms). Evoked acetylcholine release was significantly enhanced when the vagus was excited 7, 19 and 50 ms after the sympathetic nerves but it was unaltered at the 132 or 350 ms intervals, and when both nerves were stimulated simultaneously. Noradrenaline release was similar (about 6 ng per stimulation period) in all experimental groups. When sympathetic nerve stimulation had little effect in releasing noradrenaline (< 2.0 ng per stimulation period), facilitation of acetylcholine release at the 19 ms pulse interval was absent. The resting outflow of acetylcholine was unaffected by sympathetic nerve stimulation. The experiments show a facilitation of evoked acetylcholine release by sympathetic activity. As revealed by the pulse-to-pulse method this effect is confined to a relatively brief interval immediately following the excitation of the noradrenergic terminal, and is unlikely to be mimicked by exogenous drug application.