A cannulation technique was developed to collect blood samples from the ovarian vein of mares over an extended period. Ovarian venous cannulae placed in 4 mares remained patent for a mean (+/- SEM) duration of 36.8 (+/- 6.2) days. During mid-diestrus, concentrations of progesterone in the ovarian vein ipsilateral to the corpus luteum (1,663.8 +/- 238.8 ng/ml) were significantly (P less than 0.001) higher than concentrations measured in paired samples from the jugular vein (6.1 +/- 0.3 ng/ml). Concentration of estradiol in the ovarian vein ranged from a mean of 1,053.2 +/- 303.1 pg/ml during diestrus to 3,353.8 +/- 1,052.8 pg/ml during estrus, whereas values for 74% of samples collected concurrently from the jugular vein were near or below the sensitivity of the assay (10 pg/ml). Results of the study indicate that patent long-term ovarian vein cannulation can be achieved in mares. The cannulation technique provides access to important fundamental information on equine reproductive endocrinology, which to our knowledge, has not been available.