A non-surgical, non-stressful technique was used for collection of pituitary venous blood from five conscious horses every minute for two 10-min periods before and during isolation from the herd, which caused a predictable, yet humane and physiological, emotional stress. Pituitary blood was also sampled every 5 min for two approximately 90-min periods before and after isolation, while jugular blood was sampled every 15 min throughout the experiment. During isolation, all horses became agitated, hyperventilating and sweating. Packed red cell volume increased, as did pituitary venous concentrations of adrenaline (mean +/- S.E.M. concentration before isolation, 621.5 +/- 112.3 pmol/l; peak during isolation, 2665.4 +/- 869.8 pmol/l; P less than 0.05) and noradrenaline (before, 871.8 +/- 111.8 pmol/l; peak, 2726.1 +/- 547.4 pmol/l; P less than 0.02). Concentrations of arginine vasopressin (AVP) were higher in pituitary venous but not in jugular blood during isolation than during the preceding 10-min period (P less than 0.05). Although AVP secretion increased in all horses, in three of the five it rose dramatically in the first minute of isolation to 25.7 (horse 1), 13.6 (horse 4) and 145.1 (horse 5) times the level in the last sample collected before isolation. Mean pituitary venous concentrations of ACTH and alpha-MSH increased during isolation in the three horses which had large increases in AVP secretion, but, overall, stress did not significantly affect ACTH or alpha-MSH secretion. Similarly, mean jugular cortisol levels were not significantly altered by isolation. However, the magnitudes of ACTH, AVP and alpha-MSH responses to isolation were negatively correlated with the jugular cortisol level before isolation. The changes in pituitary venous concentrations of ACTH and AVP were synchronous under resting conditions, whether samples were collected at intervals of 1 (P less than 0.01) or 5 (P less than 0.005) min; however, this synchrony was lost during isolation. The changes in pituitary venous concentrations of ACTH and alpha-MSH were synchronous both at rest (P less than 0.025 for 1-min sampling, P less than 0.01 for 5-min sampling) and during isolation (P less than 0.01). We conclude that isolation stress increases AVP secretion and may alter the temporal relationship between pituitary venous concentrations of AVP and ACTH. Furthermore, the magnitude of the responses of AVP, ACTH and alpha-MSH to isolation is significantly affected by the prevailing cortisol level.