To provide further insights into non-genomic action of thyroid hormone (T3), we investigated whether Src is under control of T3 in primary calvarial osteoblasts prepared from neonatal mice. Treatment of the cells with T3 rapidly decreased SFC Y416 autophosphorylation, followed by the decrease of phosphorylated extracellular signal-regulated kinases, suggesting that T3 non-genomically suppresses Src activity. Furthermore, this T3 effect was rapid and Persistent, and was associated with the increased expression of osteocalcin (OC). To confirm the contribution of Src to the effect of T3 on OC expression, a constitutively active SFC (Y527F) was overexpressed in osteoblasts. In such cells, Y416 phosphorylation was markedly increased even in the presence of T3, and T3-dependent expression of OC was markedly attenuated. The present study demonstrates a novel, non-genomic action of T3 in primary Mouse osteoblasts, by which T3 suppresses Src thereby stimulating OC expression. (C) 2009 Elsevier Inc. All rights reserved.