To further characterize mitogenic factor(s) present in luteal extracts or luteal explant conditioned media (LCM), bovine corpora lutea (CL) were homogenized or incubated in explant culture, respectively. After evaluation of luteal extracts and LCM by using an endothelial cell proliferation bioassay, mitogenic activity was characterized by immunoneutralization with antibodies against heparin-binding (fibroblast) growth factor (HBGF) 1 or 2. LCM also were subjected to ultrafiltration, as well as anion-exchange, cation-exchange, and heparin-affinity chromatography. The presence of HBGF-2 in LCM also was evaluated by using a dot immunoblot assay. Extracts of luteal tissues and LCM stimulated (P < 0.05) proliferation of endothelial cells in a dose-dependent manner. Mitogenic activity of luteal extracts and LCM was decreased (P < 0.05) by treatment with specific antibodies against HBGF-2 or HBGF-1. LCM also contained immunoreactive HBGF-2. The mitogenic activity bound to anion exchangers, phenyl-Sepharose, and heparin–agarose, but not to cation exchangers, indicating that endothelial mitogenic activity is anionic at neutral pH, has some hydrophobic characteristics, and belongs to the HBGF family of proteins. Following ultrafiltration, endothelial mitogenic activity was retained by membranes having a 30 000 or 100 000 molecular weight cutoff. In addition, LCM was resolved into four peaks of heparin-binding endothelial mitogenic activity, each with a different affinity for heparin. These data demonstrate that bovine CL contain and produce endothelial mitogens of large molecular size, which may be important regulators of luteal function. These endothelial mitogens are heparin-binding and anionic at neutral pH. In addition, a large portion of the endothelial mitogenic activity produced by bovine CL appears to be immunologically related to HBGF-2 (basic fibroblast growth factor).Key words: angiogenesis, endothelial mitogens, corpus luteum, bovine.