Beta-blockers have beneficial effects in heart failure, although the underlying mechanism is unknown. Beta2-adrenoceptors, however, are proportionally higher in the failing human heart. This study shows several clinically used beta-blockers are agonists at the human beta2-adrenoceptor. Although these agonist effects were small at the cAMP level, they were substantial at the level of cAMP response element (CRE)-mediated gene transcription. Some of the effects of "beta-blockers" seen in heart failure may be related to the beta2-agonist actions of these compounds. CRE-gene transcription responses to beta2-agonists, forskolin, and cAMP-analogs were sensitive to p42/44-mitogen-activated protein (MAP) kinase pathway inhibitors. p42/44-MAP kinase activation was also shown directly by western blotting and enzyme-linked immunosorbent assay techniques. N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89; a protein kinase A inhibitor) stimulated cAMP accumulation and CRE gene transcription via the beta2-adrenoceptor at concentrations at which protein kinase A was inhibited, providing evidence for an alternative pathway. Propranolol, however, produced paradoxical effects; it reduced basal cAMP accumulation (via beta2-mediated inverse agonism) but stimulated beta2-mediated CRE gene transcription. This cannot be explained by a sequential pathway from Gs-adenylyl cyclase-cAMP to CRE binding protein phosphorylation. Both responses to propranolol were insensitive to pertussis toxin, thus excluding Gi-protein involvement. Propranolol CRE gene transcription responses were attenuated by p42/44-MAP kinase inhibitors and propranolol was also found to directly stimulate the p42/44-MAP kinase pathway. Studies of inositol phosphate accumulation and of protein kinase C or Rho kinase inhibitors on CRE-gene transcription provided no evidence for Gq/11 or G12/13 involvement. These data suggest that propranolol can simultaneously act as an inverse agonist through a Gs-coupled mechanism while stimulating the p42/44-MAP kinase pathway through an alternative G-protein-independent mechanism.