We have developed a non-surgical technique for long-term collection of pituitary venous blood which consists of slightly diluted hypophysial portal blood into which pituitary hormones have been secreted. In these experiments jugular and pituitary venous blood samples were collected from five unmedicated, ambulatory mares at 5-min intervals for 2-6 h on 11 occasions during the 6 days surrounding the ovulatory LH peak. Jugular blood only was collected from another five periovulatory mares without pituitary cannulae. The duration of oestrus was similar in mares with and without pituitary cannulae and all mares ovulated, showing that the procedure did not affect the reproductive axis. In all pituitary-cannulated mares the secretion of gonadotrophin-releasing hormone (GnRH), FSH and LH occurred almost continuously with broad, concurrent pulses of the three hormones superimposed upon this tonic background. Only 9% of the GnRH pulses appeared to be ineffective in inducing a rise in gonadotrophin levels. When measured in pituitary blood, gonadotrophin pulse frequency varied from 0.45 pulses/h early in the LH surge to 1.87 pulses/h at the time of ovulation. In contrast, mean pulse frequency measured in jugular blood did not exceed 1 pulse/h throughout the periovulatory period in cannulated or non-cannulated mares. The low amplitude of jugular pulses (less than 50% fractional increase) may have caused problems in identifying the pulses. In the two mares in which pituitary venous blood was sampled during more than one period before ovulation, GnRH secretion tended to be lower on the day of ovulation (day 0) than earlier in oestrus (ratio day 0:day -1; mare WV = 0.58, mare LS = 0.66), whereas LH secretion rate was higher on the day of ovulation (ratio day 0:day -1; mare WV = 1.54, mare LS = 6.68). These studies show that the painless and non-invasive collection of pituitary venous blood, which is possible only in horses, can provide a useful tool for studying hypothalamic-pituitary interactions under completely physiological conditions.