The pineal secretory product melatonin reportedly regulates release of growth hormone in humans and prevents phototherapy-induced hypocalcemia in newborn rats, suggesting that melatonin affects bone metabolism. Little is known about the effects of melatonin on bone in vitro or in vivo. The present study was undertaken to examine whether melatonin acts directly on normal human bone cells (HOB-M cells) and human osteoblastic cell line (SV-HFO cells) to affect osteogenic action in vitro. The effect of melatonin on bone cell proliferation was determined using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carbo xanilide (XTT) assay after a 24 hr incubation with melatonin. Melatonin significantly and dose-dependently increased the proliferation in HOB-M cells and SV-HFO cells by 215 +/- 22.1%, and 193 +/- 6.4%), respectively, with a maximal effect at a concentration of 50 microM. To evaluate the effect of melatonin on bone cell differentiation, alkaline phosphatase (ALP) activity, osteocalcin secretion and procollagen type I c-peptide (PICP) production (a measure of type I collagen synthesis) were measured after a 48 hr treatment. While melatonin at micromolar concentrations did not significantly affect either the ALP activity or the osteocalcin secretion, it significantly and dose-dependently increased the PICP production in HOB-M cells and SV-HFO cells by 983 +/- 42.2%, and 139 +/- 4.2%, respectively, with the maximal stimulatory doses between 50 and 100 microM. These results provide new evidence that melatonin stimulates the proliferation and type I collagen synthesis in human bone cells in vitro, suggesting that melatonin may act to stimulate bone formation.