Ca²⁺ is a key player in the regulation of many aspects of neuronal function (Berridge 1998). Changes in [Ca²⁺]_i can come from an influx of Ca²⁺ or intracellular stores. In dissociated neonatal intracardiac ganglion (ICG) neurons major sources of increases in [Ca²⁺]_i include; Ca²⁺ entry through voltage-operated Ca²⁺ channels during the action potential and nicotinic acetylcholine receptor (nAChR) channels . This increase is amplified by release of Ca²⁺ from ryanodine-sensitive Ca²⁺ stores. Activation of muscarinic ACh receptors (mAChR) also leads to Ca²⁺ release from the endoplasmic reticulum (ER) driven by a metabotropic response (Beker et al. 2003). We have investigated the [Ca²⁺]_i transients in adult rat ICG neurons evoked by somatic action potentials (direct stimulation), synaptic transmission evoked by vagal nerve stimulation (indirect) and focal application of ACh. An isolated, in vitro, right atrial ganglionic plexus preparation was used primarily associated with regulating sinoatrial node function (Sampaio et al. 2003). Intracellular recordings were made using sharp glass microelectrodes filled with Oregon Green 488 BAPTA-1 allowing simultaneous recording of electrical properties and measurement of [Ca²⁺]_i. Signals resulting from [Ca²⁺]_i changes were expressed as the ratio of fluorescence changes over baseline fluorescence, (f-f_o)/f_o . The increase in [Ca²⁺]_i. in response to a volley of 20 action potentials (10 Hz) evoked by indirect stimulation was not significantly different from that evoked by direct stimulation (0.70± 0.37 and 0.75 ±0.33 respectively, n=4). ACh responses were reduced by the mAChR antagonist, atropine (1µM) and nearly eliminated by co-application of atropine and the nAChR antagonist, mecamylamine (10µM) [see Fig 1]. Ryanodine (10 µM) reduced nACh responses [evoked by selective activation of nAChR with short pulses (≤ 100 ms) of Ach] to ∼0.6 of control values (n=3). Removal of external Ca²⁺ blunted but did not eliminate ACh induced responses. Together these results are in agreement with the nicotinic and muscarinic ACh receptor activated [Ca²⁺]_i responses reported for dissociated neonatal ICG neurones (Beker et al. 2003). In contrast to focally applied ACh there was no slow synaptic potential or [Ca²⁺]_i changes following indirect stimulation. This may be due to a difference between junctional and extrajunctional ACh receptors in these neurones.