Effects of luteal-conditioned media (LCM) on proliferation and migration of endothelial cells were used to assess angiogenic activity of corpora lutea (CL) obtained from cows on d 100 (n = 5), 150 (n = 6), 200 (n = 6), and 250 (n = 6) of gestation. Explants of CL (200 mg) were incubated for 6 h in 3 mL of serum-free media containing no hormone, LH (1 microgram/mL), prostaglandin F2 alpha (PGF2 alpha; 3 microM), or both hormones. Media from the four stages of gestation were subjected to the following procedures: 1) ultrafiltration, 2) high-salt (3.0 M NaCl) treatment and then ultrafiltration, 3) heat treatment, 4) heparin-Sepharose affinity chromatography, 5) immunoneutralization with specific antibodies against heparin-binding growth factor (HBGF)-1 and against HBGF-2, and 6) dot immunoblot assay for HBGF-2. Fractions from the first five procedures were evaluated in the endothelial cell proliferation bioassay. In addition, progesterone concentration of LCM was determined by RIA. Across all days of gestation and hormone treatments, LCM stimulated (P less than .05) proliferation and migration of endothelial cells, but activities did not differ among stages of gestation or hormone treatments. Both mitogenic and migration-stimulating fractions seemed to have Mr greater than 100,000. The mitogenic activity fraction had an apparent Mr greater than 100,000 even after treatment with high salt and was heat-labile. This endothelial mitogen was retained on heparin-Sepharose columns and was eluted with 2.0 M NaCl. Mitogenic activity was partly neutralized (P less than .05) by antibodies against HBGF-2 but not HBGF-1. Presence of HBGF-2 in LCM was detected by dot immunoblot assay.(ABSTRACT TRUNCATED AT 250 WORDS)