Formoterol, a β₂-adrenergic agonist has been shown in ovariectomized rat models to have anabolic effects on bone. However, those studies did not determine whether the effect of formoterol was by a direct action on bone cells themselves or indirectly via anabolic action on muscle. To address the question of whether formoterol could directly affect osteoblast function we investigated the expression patterns of β-adrenergic receptors (βARs) in human osteoblast-like cells and functional coupling to gene expression. Northern blot analysis showed that βAR subtypes are expressed at different levels in the osteoblast-like cell lines TE-85, SaOS-2, MG-63, and OHS-4. β₁AR expression was found in SaOS-2, OHS-4, and TE-85, but not MG-63 cells. β₂ARs are expressed at higher levels in MG-63 cells than in TE-85 and SaOS-2 cells, but were not detected in OHS-4 cells. PCR analysis paralleled the northern blot analysis except that β₃AR expression was found in one of three human primary osteoblast cDNAs tested. β₃AR expression was not found in any of the osteoblast-like cell lines. The nonspecific βAR agonist, isoproterenol, and the β₂AR-specific agonist, formoterol, induced c-fos gene expression in cultured SaOS-2 cells in an immediate early fashion. This effect was inhibited by the β₂AR-specific antagonist, ICI 118551, but not by the β₁AR-specific antagonist, CGP 20712, indicating that induction of c-fos gene expression is specifically mediated by β₂ARs. c-fos gene expression was induced by both isoproterenol and formoterol via increases in cAMP, which in turn activated the cAMP/PKA pathway; the PKA inhibitor, H89, inhibited c-fos gene expression. Thus, βARs are expressed in osteoblast-like cells and are coupled to c-fos gene expression via the β₂AR, increases in cAMP levels and activation of a PKA-dependent pathway.