Thyroid hormones L-thyroxine (T₄) and 3,5,3'-triiodo-L-thyronine (T₃) have been shown to initiate short- and long-term effects via a plasma membrane receptor site located on integrin αvβ3. Also insulin-like growth factor-1 (IGF-1) activity is known to be subject to regulation by this integrin. To investigate the possible cross-talk between T₄ and IGF-1 in rat L6 myoblasts, we have examined integrin αvβ3-mediated modulatory actions of T₄ on glucose uptake, measured through carrier-mediated 2-deoxy-[³H]-D-glucose uptake, and on cell proliferation stimulated by IGF-1, assessed by cell counting, [³H]-thymidine incorporation and FACS analysis. IGF-1 stimulated glucose transport and cell proliferation via the cell surface IGF-1 receptor (IGF1R) and, downstream of the receptor, by the phosphatidylinositol 3-kinase signal transduction pathway. Addition of 0.1 nM free T₄ caused little or no cell proliferation, but prevented both glucose uptake and proliferative actions of IGF1. These actions of T₄ were mediated by an Arg-Gly-Asp (RGD)-sensitive pathway, suggesting the existence of crosstalk between IGF1R and the T₄ receptor located near the RGD recognition site on the integrin. An RGD-sequence-containing integrin inhibitor, a monoclonal antibody to αvβ3, and the T₄ metabolite tetraiodothyroacetic acid all blocked the inhibition by T₄ of IGF-1-stimulated glucose uptake and cell proliferation. Western blotting confirmed roles for activated phosphatidylinositol 3-kinase and extracellular regulated kinase 1/2 (ERK1/2) in the effects of IGF-1, and also showed a role for ERK1/2 in the actions of T₄ that modified the effects of IGF-1. We conclude that thyroid hormone inhibits IGF-1-stimulated glucose uptake and cell proliferation in L6 myoblasts.