Using membrane fractions (MF) from guinea pig alveolar macrophages (AM), we investigated the effects of sensitization and antigen challenge on the stepwise activation of adenylyl cyclase considering receptor binding, G-protein coupling and direct stimulation of the enzyme. Receptor binding studies, using [125I]ICYP as the beta-adrenoceptor specific ligand, show that neither receptor number (Bmax) nor receptor affinity constants (Kd values) were affected by sensitization or antigen challenge. Using forskolin as a direct stimulant of AC, alterations in the enzymatic activity of AC could be excluded. Pretreatment of the different MF with cholera toxin (CT, a toxin which eliminates GTPase activity) and subsequent stimulation of AC with GTP, shows an increased responsiveness in MF from sensitized and antigen challenged AM. In addition, pretreatment of MF from naive AM with increasing doses of CT results in a maximal AC response at the higher concentrations used (50-100 micrograms/mL), an effect not observed in MF from sensitized and antigen challenged AM. In these MF, the AC response still increases after pretreatment with such doses of CT. These data suggest that the enhanced AC responsiveness in AM, induced by sensitization and antigen challenge, results from alterations in alpha s-subunits.