The autonomic nervous system of mammals displays extensive neurotransmitter diversity. The guinea-pig sympathetic nervous system has served as a model for in vivo studies of neurotransmitter co-expression. We have developed methods for the dissociation and long-term culture of adult guinea-pig prevertebral sympathetic ganglia. The neurotransmitter properties of cultured adult guinea-pig sympathetic neurons from the celiac and superior mesenteric ganglia were examined. Cultured principal neurons were found to display many of their in vivo neurotransmitter characteristics, including catecholamine-specific histofluorescence and immunoreactivity for tyrosine hydroxylase and the neuropeptides, neuropeptide Y, somatostatin and vasoactive intestinal polypeptide. In addition, the cultures of both ganglia displayed the various neurotransmitter characteristics in approximately the same percentage of the cultured neurons as reported in in vivo studies. A small percentage of principal neurons and many small, intensely fluorescent-like cells labeled with antibodies against 5-hydroxytryptamine. Many of the principal neurons were found to bear 5-hydroxytryptamine3 receptors, suggesting a possible role for this neurotransmitter in neuron-neuron and small, intensely fluorescent cell-neuron transmission. We conclude that adult guinea-pig sympathetic neurons retain their neurotransmitter phenotypes when grown in dissociated cell culture. These properties include the co-expression of the classical transmitters, norepinephrine, 5-hydroxytryptamine and neuropeptides. This culture preparation will prove to be valuable in future studies on the functional properties of these neurons and their development.