A highly specific heterologous double-antibody RIA [radioimmunoassay] was developed to measure rabbit PRL [prolactin] by using guinea pig antiserum to human PRL and ovine [125I]iodo-PRL. Rabbit pituitary PRL and serum gave parallel dose-response curves in the assay and no cross-reaction (< 0.1%) occurred with GH [growth hormone], placental lactogens, LH [luteinizing hormone], FSH [follicle stimulating hormone] or TSH [thyrotropin] from several different species. The assay is suitable for the measurement of human, ovine, bovine, caprine and canine PRL in addition to rabbit PRL, but shows no cross-reaction with rat PRL. Reproducibility and precision of the assay are within acceptable limits. Gel filtration of rabbit pituitary PRL and rabbit serum on Sephadex G-100 revealed coincident peaks of activity measured by RIA and by PRL radioreceptor assay. The MW of rabbit PRL appeared similar to that of ovine PRL. Serum PRL levels increased after the injection of both TRH [thyrotropin releasing hormone]and chlorpromazine and were reduced by CB154 (bromocriptine). Venepuncture stress caused an increase in PRL in nonpregnant or postpartum non-suckled animals, but small or no increases were seen in lactating female rabbits.