In the paraventricular nucleus (PVN) of the hypothalamus, nitric oxide (NO) inhibits sympathetic outflow through increased GABA release. However, the signal transduction pathways involved in its action remain unclear. In the present study, we determined the role of cGMP, soluble guanylyl cyclase, and protein kinase G in the potentiating effect of NO on synaptic GABA release to spinally projecting PVN neurons. The PVN neurons were retrogradely labeled by a fluorescent tracer injected into the thoracic spinal cord of rats. Whole-cell voltage-clamp recordings were performed on labeled PVN neurons in the hypothalamic slice. Bath application of the NO donor, S-nitroso-N-acetyl- penicillamine (SNAP), reproducibly increased the frequency of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) without changing the amplitude and the decay time constant. Neither replacement of Ca++ with Co++ nor application of Cd++ to block the Ca++ channel altered the effect of SNAP on mIPSCs. Also, the effect of SNAP on mIPSCs was not significantly affected by thapsigargin, a Ca++-ATPase inhibitor that depletes intracellular Ca++ stores. Application of a membrane- permeant cGMP analog, pCPT-cGMP, mimicked the effect of SNAP on mIPSCs in the presence of a phosphodiesterase inhibitor, IBMX. Furthermore, both the soluble guanylyl cyclase inhibitor, ODQ, and the specific protein kinase G inhibitor, Rp-pCPT-cGMP, abolished the effect of SNAP on mIPSCs. Thus, these data provide substantial new information that NO potentiates GABAergic synaptic inputs to spinally projecting PVN neurons through a cGMP- protein kinase G pathway.