A number of factors have been shown to influence osteoblastic proliferation, including fluoride. Recent observations suggest that heterotrimeric G proteins are probably involved in the mitogenic response induced by this agent, further suggesting a role of guanosine 5'-triphosphate (GTP)-binding protein-coupled receptors (GPCR) in the regulation of osteoblastic cell growth. We therefore explored what mitogenic factors known to activate GPCR can influence the replication of mouse osteoblast-like MC3T3-E1 cells. Among several candidates, epinephrine was found to be a potent mitogen for these cells, and its effect on the growth and differentiation of these cells was further investigated. Deoxyribonucleic acid (DNA) synthesis was dose dependently enhanced by this catecholamine in the concentration range of 1 nmol/L-10 micromol/L. Stimulation of DNA synthesis by catecholamines was in the order of epinephrine > norepinephrine >> isoproterenol, indicating that alpha adrenergic receptors mediated this cellular response. Further analysis with specific adrenergic receptor agonists and antagonists suggested that the mitogenic response induced by epinephrine in MC3T3-E1 cells is mediated by alpha1 adrenergic receptors. In addition to its effect on cell replication, epinephrine also enhanced alkaline phosphatase (ALP) activity in these cells but had little effect on collagen synthesis and osteocalcin production. As for the mitogenic response, the change in ALP activity was found to be mediated by alpha1 adrenergic receptors. Both effects of epinephrine on cell replication and ALP activity were markedly reduced by pretreatment of the cells with pertussis toxin (PTX), suggesting a role of Gi proteins. These effects were also completely blocked by pretreatment of the cells with 50 micromol/L genistein, a nonselective inhibitor of tyrosine kinase. In conclusion, the results indicate that epinephrine enhances replication and ALP activity of MC3T3-E1 osteoblast-like cells via alpha1 adrenergic receptors coupled to Gi proteins. The signaling mechanism probably involves a tyrosine phosphorylation mechanism. These observations suggest that PTX-sensitive G proteins are potent mediators of cell proliferation and ALP activity of osteoblast-like cells in response to factors acting through G protein-coupled receptors.