Physiological properties of the synapses formed betweeen explants of spinal cord and dissociated autonomic ganglion neurons in tissue culture were studied using intracellular and extracellular stimulation and recording techniques (as well as iontophoresis) with a culture perfusion system allowing continuous microscopic observation during repeated changes of the bathing medium. The principal neurons of the superior cervical ganglion (SCGN) were dissociated from perinatal rats and the spinal cord explants were obtained from 15-day rat fetuses; these were allowed to mature for 3-10 weeks in co-culture. Recordings from over 1000 SCGN established that: (a) spontaneous small depolarizations and action potentials occurred in 20% of the SCGN studied, (b) the EPSPs observed in SCGN after spinal cord stimulation were sensitive to decreased Ca2+ and increased Mg2+, as well as to d-tubocurare, hexamethonium and mecamylamine, but not to atropine (at 10-6 M concentration) or to the alpha-adrenergic blocking agents phentolamine or phenoxybenzamine; no potentiation of the EPSPs was seen with neostigmine or eserine, (c) acetylcholine directly applied to the SCGN was seen to mimic the responses seen after spinal cord stimulation; tetrodotoxin blocked both direct and iontophoretically fired action potentials, with only a suprathreshold acetylcholine potential remaining. These synapses were not sensitive to α-bungarotoxin. It is concluded that the synapses formed by spinal cord neurites on principal SCGN in tissue culture are nicotinic cholinergic, and that removal of the suprasegmental inputs in an analogous fashion to the fibrillation seen in denervated muscle fibers72. Alternatively, the spinal cord tissue may be more active due to the loss of supraspinal inhibitory influences8. The source of spontaneous synaptic activity observed in SCGN cultures after removal of the spinal cord explants or in pure SCGN networks64 (Wakshull and Burton, unpublished observations) must be propagated synaptic activity from other sympathetic neurons11,46,64. The origin of this activity may be intrinsic pacemaker-like responses in some SCGN. Direct recording of these intrinsic responses has not been obtained even in reduced Ca2+ medium, although this possibility still cannot be ruled out. No spontaneous potentials were noted in denervated frog41 and mammalian sympathetic ganglia54,56 but reinnervation, which occurs in culture, may impart some unknown instability to these neurons. Alternatively, some changes in the composition or flow of the medium may activate a response that then propagates through the synaptic networks in the culture eventually reaching the recorded neuron. This suggestion is tenable provided that the SCGN are synaptically connected in vitro to a great extent46.