Beta-agonists, through activation of the beta(2)-adrenergic receptor (beta(2)AR)-G(s)-adenylyl cyclase (AC) pathway, promote bronchodilation via functional antagonism of airway smooth muscle (ASM) spasmogens associated with the asthmatic state. Although previous studies have demonstrated that beta(2)AR signaling in ASM is subject to homologous (beta-agonist-induced) beta(2)AR desensitization, the potential for inflammatory and contractile agents to impact beta(2)AR signaling in ASM through heterologous mechanisms has not been defined. Here we report that chronic exposure of human ASM (HASM) to carbachol, serotonin, the thromboxane analogue U46619, or histamine induced little change or a small increase in isoproterenol-stimulated cyclic adenosine monophosphate (cAMP) formation, but significantly increased cAMP formation elicited by stimulation with forskolin. This latter increase in intrinsic AC activity was largely reversed by pertussis toxin pretreatment, and was unaffected by protein kinase C inhibition. Analysis of both AC function and isoform expression supports a dominant role of AC VI in HASM, and points to important differences in ASM AC isoform expression among species. Additional studies identify AC as the limiting component in beta(2)AR-G(s)-AC signaling in HASM, and thus a potentially important target of therapeutic strategies designed to influence airway contractile state.