The secretion of cortisol increases in awake dogs after small hemorrhage, with little or no change in plasma immunoreactive (IR) ACTH. To determined if IR-ACTH reflects bioactive (B) ACTH after hemorrhage, changes in BACTH and IR-ACTH were determined in awake dogs. Trained dogs were prepared with adrenal venous and femoral arterial and venous cannula. Experiments performed 48-96 h after surgery consisted of blood sampling from the adrenal vein and femoral artery before and after a 7.5-10 ml/kg hemorrhage done over 3 min. Cortisol was assayed by HPLC with UV detection, and adrenal secretory rates were calculated using adrenal blood flow. Arterial B-ACTH was assayed after extraction using rat adrenal cells dispersed with collagenase; corticosterone produced was assayed by HPLC-UV. Arterial IR-ACTH was assayed by RIA using antisera directed against ACTH-(l-24). Dogs in which hypotension occurred (change in mean arterial pressure, -31 ± 3; n = 6) were compared to dogs in which mean arterial pressure did not change (change in mean arterial pressure, - 6 ± 2; n = 5). In the absence of hypotension, B-ACTH increased by 10 min after the onset of hemorrhage coincident with increased cortisol secretion, whereas IR-ACTH did not increase until 20 min. Resting IR-ACTH was greater than B-ACTH (11 ± 1 vs. 2 ± 1 pg/ml), but the peak response of B-ACTH was greater than that of IR-ACTH (13 ± 4 vs. 8 ± 3 pg/ml). In the presence of hypotension, B-ACTH increased by 4 min, and IRACTH and cortisol secretion increased by 8 min after the onset of hemorrhage. Resting IR-ACTH was greater than B-ACTH (27 ± 5 vs. 6 ± 1 pg/ml), and the peak response of B-ACTH was less than that of IR-ACTH (64 ± 26 vs. 112 ± 33 pg/ml). In dogs subjected to blood sampling without hemorrhage (n = 4), resting IR-ACTH was greater than B-ACTH (34 ± 5 vs. 5 ± 1 pg/ml), but there was no change in B-ACTH, IR-ACTH, or cortisol secretion. The results show that small hemorrhage elicits changes in B-ACTH that are dissociated in time and magnitude from changes in IR-ACTH, are coincident with changes in cortisol secretion, and are greater in dogs that fail to maintain arterial pressure. These data indicate that B-ACTH predicts more accurately the change in cortisol secretion than does IRACTH after small hemorrhage.