We investigated the effects of hypergravity on DNA synthesis and alkaline phosphatase (ALP) activity in cloned osteoblast-like cells, MC3T3-E1. Hypergravity (5 x g) stimulated DNA synthesis in these cells in a time-dependent manner and increased it approximately up to 150% of that of the control (1 x g). 12-O-Tetra-decanoylphorbol-13-acetate (TPA), a protein kinase C activator, and insulin-like growth factor I (IGF-I) enhanced DNA synthesis additively with hypergravity (5 x g). An increase in ALP activity induced by 10% fetal calf serum (FCS) was suppressed by hypergravity (2 x g, 5 x g). Five x g completely suppressed the increase in ALP activity. TPA and hypergravity (2 x g) suppressed the increase in ALP activity induced by FCS additively. Hypergravity (5 x g) showed no significant effect on cAMP nor cGMP production in these cells, but increased prostaglandin E2 (PGE2) production. Exogenous PGE2 stimulated DNA synthesis in these cells but had little effect on 10% FCS-induced ALP activity. These results suggest that hypergravity stimulates proliferation but suppresses differentiation of osteoblast-like cells through a pathway independent of the activation of protein kinase C and the production of cyclic nucleotides, and that hypergravity and IGF-I stimulate proliferation of these cells through an independent signal transduction pathway. Moreover, our data strongly suggest that PGE2 mediates the signalling of hypergravity on the proliferation of osteoblast-like cells.