Mechanisms by which beta-adrenergic receptor (AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that AR agonists would inhibit mitogenesis in HASM cells via the ₂AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [³H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The non-selective AR agonist isoproterenol and the ₂AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A ₂AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a ₁AR-selective antagonist, confirming ₂AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE₂ and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. ₂AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA), as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 µM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 µM). These data show that ₂AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacologic evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.