Radioimmunoassay methods were employed to quantitatively characterize secretion of the avian antidiuretic hormone [arginine vasotocin (AVT)] by the hypothalamo-neurohypophyseal system (HNS) of the conscious domestic fowl in response to chronic dehydration. Water deprivation permitted characterization of AVT secretion in response to the combined stimuli of extracellular hyperosmolality and hypovolemia; the subsequent repletion of extracellular volume permitted separation of potential osmotic and volemic factors involved in the regulation of AVT secretion. In normally hydrated birds, plasma AVT (PAVT) and plasma osmolality (Posm) averaged 2.2 +/- 0.3 microU/ml (10.5 +/- 1.4 pg/ml) and 309.3 +/- 0.7 mosmol/kg H2O, respectively (means +/- SE). With water deprivation, PAVT and Posm of the birds increased in parallel in a curvilinear manner to maxima of 13.1 +/- 0.6 microU/ml (62.4 +/- 2.9 pg/ml) and 346.6 +/- 2.0 mosmol/kg H2O, respectively, at 96 h of dehydration. The isosmotic repletion of extracellular volume at 96 h by acute intravenous infusion failed to alter 96-h PAVT values. The results indicate that AVT secretion is closely linked to the state of hydration during negative fluid balance in the domestic fowl. Analysis of the data indicated that increases in PAVT that occur with dehydration are mediated primarily by extracellular hyperosmolality and that the HNS of the domestic fowl is relatively insensitive to the simultaneous hypovolemia incurred with fluid deprivation.