A homologous radioimmunoassay for chicken prolactin is described. The assay is based on recombinant-derived chicken prolactin that has been used to raise an antibody and produce 125I-labeled tracer and assay standards. The radioimmunoassay measures a minimum of 0.03 +/- 0.01 ng of the recombinant prolactin with 50% displacement of binding by 0.6 +/- 0.1 ng. Chicken plasma and pituitary immunoreactivity dilute in parallel with the standard curve. The mean (+/- SE) concentrations of plasma prolactin were 12.1 +/- 1.5, 433.2 +/- 5.5, and 1609.8 +/- 18.5 ng/ml in out-of-lay, laying, and incubating bantam hens, respectively. The binding of recombinant prolactin was not displaced by extracts of neural lobe or plasma from a hypophysectomized hen. Plasma prolactin concentrations were increased after intravenous administration of chicken vasoactive intestinal polypeptide or quipazine, a 5-HT agonist. The assay measures immunoreactive prolactin in other birds. Plasma from quail, turkey, great tit, and wryneck displaced the 125I-labeled prolactin tracer parallel to the standard curve. Starling, ring dove, and penguin plasma displaced the binding in a nonparallel manner.