Pancreatic neurons receive and integrate synaptic input from a wide variety of extrinsic nerves while providing the predominant innervation of pancreatic acini, ducts, and islets of Langerhans. Here we report the first primary cultures of adult rabbit pancreatic neurons, isolated from extrinsic nerves and secretory cells, and evaluate the neurochemical and electrical properties of these neurons. Pancreatic cultures consisted of single and clustered neurons, extended varicose processes after 3-4 days in culture, and formed interconnecting networks of neurons after 7-10 days. Isolated pancreatic islet cells, added to established neuron cultures, remained attached and viable for several weeks and received innervation by varicose nerve fibers. Histochemical staining revealed populations of neurons positive for acetylcholinesterase (75%), NADPH-diaphorase (62%), nitric oxide synthase (73%), and/or vasoactive intestinal peptide (VIP) (65%). Intracellular recordings revealed active and passive electrical properties comparable to those of neurons from intact ganglia. Several distinct populations of neurons were identified by their firing patterns (phasic vs. tonic) in response to prolonged depolarizing currents or the amplitude and duration of their after-spike hyperpolarizations. Low-amplitude, pacemaker-like potentials were observed in 25% of the neurons and, in older cultures with extensive networks of fibers, spontaneous fast excitatory postsynaptic potentials (EP-SPs) also occurred. Thus these cultures retained the salient neurochemical and electrophysiologic properties observed in pancreatic neurons from intact ganglia and offer a good model for studies of the intrinsic innervation of the pancreas.