Thrombospondin is a 450 kDa glycoprotein first identified as a secretory product of thrombin-stimulated platelets. Recently, components of the thrombin-generating pathway of hemostasis have been identified in follicular fluid. Endogenously synthesized thrombospondin is found in the extracellular matrix of hormone responsive cells, such as bovine adrenocortical cells, and has been demonstrated to be influential in regulating their morphology. Western blot analysis of follicular fluid and granulosa cell lysates, isolated from bovine ovaries collected at slaughter, detected the presence of thrombospondin. Northern blot analysis of total RNA from granulosa cells demonstrated that thrombospondin 1 and thrombospondin 2 mRNA is present at highest levels in the early stages of follicular organization. These studies reveal that thrombospondin is produced by granulosa cells and secreted into the surrounding follicular fluid in decreasing concentrations as the follicle size increases, supporting a potential role for thrombospondin in bovine follicular development.