The human neuroblastoma cell line SK-N-SH has been used as a model system to study the interactions of the human estrogen receptor (hER) with neurotransmitters. We have successfully transfected these cells using an adenoviral delivery system and have reconstituted ligand-dependent responses to estradiol and ligand-independent responses to a series of dopamine D1 receptor agonists. The full agonist for the D1 receptor, SKF 82958, shows a robust activation of hER, comparable to that induced by estradiol. This activation is blocked by the protein kinase A inhibitor H-89, is mimicked by forskolin, and is therefore thought to be mediated in part through the cAMP/protein kinase A pathway. We have examined deletion mutants of hER for activation by SKF 82958 and find that both its transactivation domains, AF-1 and AF-2, must cooperate to impart the full response to the agonist. Significantly, an agonist of the muscarinic acetylcholine receptor, carbachol, though not active by itself, synergistically activates hER in conjunction with suboptimal doses of SKF 82958. This is the first reported instance of two neurotransmitters synergizing to activate a member of the nuclear receptor superfamily, and might predict a role for multiple neural inputs modulating the effects of these receptors in the central nervous system.