Although the acceleration of bone regeneration by laser treatment has been reported, the mechanisms of action of laser on bone are unclear. To determine the target cells responsible for the action of laser irradiation and roles of irradiation on these cells during bone formation, we investigated the effects of low-energy laser irradiation at various cell culture stages on cellular proliferation, bone nodule formation, alkaline phosphatase activity, and osteocalcin gene expression, employing rat calvarial cells. Osteoblast-like cells isolated from fetal rat calvariae were irradiated once with a low-energy Ga-Al-As laser (830 nm, 500 mW) at various cell culture stages (days 1-16). Laser irradiation at early stages of culture significantly stimulated cellular proliferation, ALP activity, and osteocalcin gene expression thereafter. Furthermore, laser irradiation at earlier stages of culture significantly stimulated a greater number (1.7-fold) and larger area (3.4-fold) of bone nodules that had developed in the culture dish on day 21. However, these effects could not be found by irradiation at a later date. These results suggest that laser irradiation may play two principal roles in stimulating bone formation. One is stimulation of cellular proliferation, especially proliferation of nodule-forming cells of osteoblast lineage, and the other is stimulation of cellular differentiation, especially to committed precursors, resulting in an increase in the number of more differentiated osteoblastic cells and an increase in bone formation. Both bone-formation-stimulating roles may be exhibited by laser irradiation to immature cells only.