The acute effects of nicotine [1-methyl-2-(3-pyridyl)pyrrolidine] on the formation and resorption of bone were examined in cultures of clonal rat calvarial osteogenic cells (ROB-C26) and clonal mouse calvarial preosteoblastic cells (MC3T3-E1), as well as in osteoclast-like cells formed during coculture of mouse bone marrow cells and clonal stromal cells from mouse bone marrow, ST2 cells, at concentrations that occur in the saliva of smokeless tobacco users. Nicotine stimulated the rate of deposition of Ca(2+) by ROB-C26 cells, as well as the alkaline phosphatase activity of these cells, in a dose-dependent manner. However, both activities decreased in MC3T3-E1 cells that had been exposed to nicotine. These results indicate that nicotine affected osteoblastic differentiation in osteoblast-like cells. By contrast, nicotine reduced, in a dose-dependent manner, the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the formation of pits on slices of dentine, both of which are typical characteristics of osteoclasts. Our results suggest that nicotine might have critical effects on bone metabolism.