The expression of three enteric neuropeptides was examined in freshly dispersed ganglia and in ganglia cultured for up to 28 days. During culture, glial cells grew into a flat sheet surrounding a cluster of neurons identified with neuron-specific enolase (13 +/- 2/ganglion), which remained constant throughout the period of culture. The neurons underwent a distinctive temporal change, resulting in overexpression of substance P (SP), normal expression of somatostatin, and virtual suppression of vasoactive intestinal peptide (VIP). Three weeks after the start of culture, the ganglia contained and released (in response to 55 nM KCl, 0.1 mM 1,1-dimethyl-4-phenylpiperazinium, or 1 microM gastrin-releasing peptide) twice as much SP as freshly dispersed ganglia, corresponding to a sevenfold increase per cultured neuron; content and release of somatostatin did not change. SP content and release declined to 1.5% of those found in control cultures when nonneuronal cells were suppressed with cytosine arabinoside but were partially restored (13-17% of control) by nerve growth factor. In marked contrast, VIP was minimally (< 1%) present in and released from ganglia after the third day in culture. Suppression of VIP could reflect a selective loss of VIP neurons and/or VIP expression.