Our latest data show that nitric oxide (NO) potentates GABAergic transmission via an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the nucleus tractus solitarii (NTS) GABAergic interneurons (Wang et al. 2006). However, the mechanism mediating NO action remains unclear. In sea urchin eggs NO regulated calcium homeostasis via cyclic ADP ribose (cADPR)/ryanodine-sensitive stores (Galione et al. 1993). We hypothesised that NO-stimulated potentiation of GABAergic transmission also may be mediated by a cADPR-dependent mechanism. Organotypic rat brainstem slice cultures were transfected with adenoviral vector containing 3.7 kb of the GAD67 promoter driving expression of enhanced green fluorescent protein. Fluorescent GABAergic neurons were visualised by combined application of confocal and DIC optics and recorded in whole-cell patch clamp mode. The intracellular solution contained the red-shifted calcium indicator Rhod-2. The ratio of fluorescence intensity (F/F₀) was used to assess changes in [Ca²⁺]_i. Following bath application of 10 μM DEA/NO, [Ca²⁺]_i clearly increased in somata (+20 ± 4%, n=8/9, P<0.01) and dendrites (+30 ± 4%, n=9, P< 0.001), but the most dramatic increase occurred in the putative axons (+40 ± 10%, n=5/6, P<0.05). The soluble guanylate cyclase inhibitor ODQ blocked DEA/NO action in 6 of 7 neurons tested. An antagonist of cADPR receptors, 8-Br-cADPR, introduced via patch pipette (100 μM in the patch pipette solution), almost completely abolished DEA/NO-induced [Ca²⁺]_i increases in 7 of 8 neurons. To test whether cADPR also mediates NO-induced potentiation of GABAergic postsynaptic inhibitory potentials (IPSPs) whole-cell patch clamp recordings were performed from NTS neurons in acutely prepared brainstem slices. Monosynaptic IPSPs were evoked by electrical stimulation within the medial NTS in the presence of CNQX (20 μM). 1 μM DEA/NO (~55 nM of free NO) reversibly increased the amplitude of monosynaptic IPSPs by ~32% (n=5, P<0.01). Pre-treatment with 30 μM 8-Br-cADPR essentially abolished DEA/NO-induced IPSP potentiation (n=5). Therefore, the sGC/cGMP signalling cascade may lead to activation of ADP-ribosyl cyclase via cGMP-dependent protein phosphorylation and subsequent production of cADPR. This causes sensitisation of cADPR/ryanodine sensitive stores which boost action-potential-mediated Ca2+ release and promotes GABA exocytosis, resulting in potentiation of GABA-mediated IPSPs. In summary, NO potentiates GABAergic transmission via an evolutionary conserved cGMP/cADPR/ryanodine signalling pathway.