It is well known that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p44/p42 mitogen-activated protein (MAP) kinase, which limits T(3)-induced alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p44/p42 MAP kinase or p38 MAP kinase is involved in the thyroid hormone-stimulated osteocalcin synthesis in these cells. T(3) markedly induced the phosphorylation of p38 MAP kinase in addition to p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the T(3)-induced synthesis of osteocalcin. On the contrary, the T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 and PD169316, inhibitors of p38 MAP kinase. SB203580, PD169316 or PD98059 suppressed the T(3)-phosphorylation of myelin basic protein. T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 or PD169316 also in primary cultured mouse osteoblasts. These results strongly suggest that p38 MAP kinase but not p44/p42 MAP kinase takes part in the thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.