Benzo[a]pyrene regulates osteoblast proliferation through an estrogen receptor-related cyclooxygenase-2 pathway. Polycyclic aromatic hydrocarbons (PAHs) have been known as a kind of xenoestrogen. Benzo[a]pyrene, a PAH present in tobacco smoke and tar, has been implicated in the induction of cell proliferation as well as tumors including osteosarcoma. Nevertheless, the literature about the action of benzo[a]pyrene on the bone system is rare. It has been identified that osteoblasts owned the estrogen receptors and estrogen could modulate the osteoblast proliferation. In this study, we found that benzo[a]pyrene was capable of increasing the cell proliferation in cultured rat osteoblasts, human osteosarcoma cell line (MG-63), and estrogen sensitive human cell line (MCF-7) but not in the human estrogen receptor negative cell line (MDA-MB-231). This benzo[a]pyrene-induced osteoblast proliferation could be inhibited by the estrogen receptor antagonist ICI182780 and tamoxifen, PD98059 [extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3K) inhibitor] but not alpha-naphthoflavone (aryl hydrocarbon receptor antagonist) and SB203580 (p38 MAPK inhibitor). Western blot analysis showed that benzo[a]pyrene could induce the phosphorylation of ERK1/2 and Akt (PI3K downstream effector) in osteoblasts. The proliferating cell nuclear antigen protein levels in nuclear fraction of osteoblasts were also increased by benzo[a]pyrene. Moreover, cyclooxygenase-2 (COX-2), but not COX-1, expression could be induced in osteoblasts under benzo[a]pyrene treatment. Its upregulation was associated with the induction of prostaglandin E(2) (PGE(2)). COX-2 inhibitors NS398 and aspirin are capable of inhibiting the benzo[a]pyrene-induced osteoblast proliferation. These results indicate that benzo[a]pyrene may modulate the osteoblast proliferation through activation of COX-2 protein.