A highly specific heterologous double-antibody RIA has been developed to measure rabbit PRL by using guinea pig antiserum to human PRL and ovine [125 I]iodo-PRL. Rabbit pituitary PRL and serum give parallel dose-response curves in the assay and no cross-reaction (less than 0.1%) occurs with GH, placental lactogens, LH, FSH, or TSH 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 molecular weight of rabbit PRL appeared similar to that of ovine PRL. Serum PRL levels increased after the injection of both TRH 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.