A RIA was developed for the measurement of immunoreactive atrial natriuretic factor (irANF) in avian plasma and was used to investigate the relationship between vascular volume and plasma irANF concentrations in conscious Pekin ducks. In normally hydrated birds, the mean plasma irANF concentration was 78.5 +/- 6.8 pg/ml. Blood volume expansion by iv infusion of isotonic saline at 1 ml/min elevated irANF concentrations by 132% after 1 h and by 233% after 2 h. Reduction of the vascular volume by nonhypotensive hemorrhage of 10% and then 20% of the total blood volume reduced irANF levels by 16% and 42%, respectively, 5 min after blood removal was completed. Similarly, in birds deprived of water for 24 h and with blood volume reduced by 4.8%, plasma irANF concentrations were 50% lower than in the same euhydrated animals. The observed correlation between plasma irANF concentrations and vascular volume is consistent with the concept that ANF has a physiological role in avian volume homeostasis.