We have examined the interaction between muscarinic and ₂-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca²⁺ concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca²⁺ and IP formation, activation of ₂-receptors does not significantly increase either intracellular Ca²⁺ or IPs over basal levels. However, simultaneous activation of muscarinic and ₂-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca²⁺ increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and ₂-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca²⁺, because it is decreased but not prevented by blocking the entry of Ca²⁺ with LaCl₃ or chelating intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca²⁺ resulting from simultaneous activation of muscarinic and ₂-adrenergic receptors.