The effects of thyroid hormone analogues on modulation of angiogenesis have been studied in the chick chorioallantoic membrane model. Generation of new blood vessels from existing vessels was increased 3-fold by either l-thyroxine (T4; 10(-7) mol/L) or 3,5,3'-triiodo-l-thyronine (10(-9) mol/L). T4-agarose reproduced the effects of T4, and tetraiodothyroacetic acid (tetrac) inhibited the effects of both T4 and T4-agarose. Tetrac itself was inactive and is known to block actions of T4 on signal transduction that are initiated at the plasma membrane. T4 and basic fibroblast growth factor (FGF2) were comparably effective as inducers of angiogenesis. Low concentrations of FGF2 combined with submaximal concentrations of T4 produced an additive angiogenic response. Anti-FGF2 inhibited the angiogenic effect of T4. The proangiogenic effects of T4 and FGF2 were blocked by PD 98059, a mitogen-activated protein kinase (MAPK) pathway inhibitor. Endothelial cells (ECV304) treated with T4 or FGF2 for 15 minutes demonstrated activation of MAPK, an effect inhibited by PD 98059 and the protein kinase C inhibitor CGP41251. Reverse transcription-polymerase chain reaction of RNA extracted from endothelial cells treated with T4 revealed increased abundance of FGF2 transcript at 6 to 48 hours, and after 72 hours, the medium of treated cells showed increased FGF2 content, an effect inhibited by PD 98059. Thus, thyroid hormone is shown to be a proangiogenic factor. This action, initiated at the plasma membrane, is MAPK dependent and mediated by FGF2.