Previous investigations have established that electrical activity or chronic depolarization influences the development of neonatal rat sympathetic neurons in dissociated cell culture. Depolarization reduces their ability to respond to a cholinergic inducing factor produced by non-neuronal cells, allowing normal adrenergic differentiation to proceed (Walicke, P., R. Campenot, and P. Patterson (1977) Proc. Natl. Acad. Sci. U. S. A. 74: 5767–5771). The present study examines whether the developmental effects of depolarization are mediated through cyclic nucleotides. Addition of dibutyryl cAMP, dibutyryl cGMP, adenosine, prostaglandin E1, and cholera toxin all raise neuronal cyclic nucleotide levels and qualitatively mimic the developmental effects of depolarization. However, the quantitative decrease in acetylcholine production caused by these cyclic nucleotide agents is much smaller than that caused by depolarization. Short (48-hr) exposures to the cyclic nucleotide derivatives do not alter transmitter synthesis, indicating that long term developmental changes are involved. Chronic depolarization with elevated K+ increases neuronal cAMP 2-fold but has little effect on cGMP. The increase in cAMP is maintained during several weeks of depolarization and is present as early as the 3rd day in vitro, preceding the significant alterations in adrenergic and cholinergic differentiation. Exposure to 2 mM theophylline also increases neuronal cAMP, but in contrast to the other agents, it enhances cholinergic differentiation. In combination with elevated Ktheophylline further increases neuronal cAMP but still favors cholinergic differentiation. Thus, although cAMP satisfies some criteria for being the second messenger in the developmental effects of depolarization, several findings are consistent with the nucleotide playing a central role: (i) Depolarization has much larger effects on transmitter choice than the cyclic nucleotide agents and (ii) theophylline can uncouple cyclic nucleotide levels from the developmental events.