3,5,3'-Triiodo-L-thyronine (T₃), but not L-thyroxine (T₄), activated phosphatidyl-inositol 3-kinase (PI 3-K) by a receptor on v3 integrin in human glioblastoma U87MG cells. We confirmed that both T₃ and T₄ activate ERK1/2, but activated ERK1/2 did not contribute to T₃-induced PI 3-K activation and an inhibitor of PI 3-K, LY294002, did not block activation of ERK1/2 by physiological concentrations of T₃ and T₄. Thus, PI 3-K and ERK1/2 are parallel signal transduction pathways in T₃-treated U87MG cells. T₃ and T₄ both caused proliferation of U87MG cells. An ERK1/2 inhibitor, PD98059, blocked the proliferative action of both iodothyronines, but LY294002 did not affect proliferation caused by T₃ and T₄. siRNA knockdown of PI 3-K confirmed that PI 3-K was not involved in the proliferative action of T₃ on U87MG cells. Shuttling of nuclear thyroid hormone receptor- (TR) from cytoplasm to nucleus was stimulated in these cells by T₃; this process was inhibited by LY294002 but not by PD98059 or ERK1/2 siRNA. In addition, hypoxia-inducible factor (HIF)-1 mRNA accumulation when induced by T₃ was PI 3-K-dependent but required ERK1/2 when induced by T₄. In summary, thyroid hormone nongenomically activates the ERK1/2 and PI 3-K pathways via the hormone receptor on integrin v3 in U87MG cells, although only T₃ activates PI 3-K. PI 3-K does not mediate cell proliferation. T₃ interacts with the integrin receptor by two mechanisms, one of which is proliferative and independent of PI 3-K and the second of which causes PI 3-K activation-dependent trafficking of TR from cytosol to the nucleus of T₃-treated cells.