Antibodies raised in guinea pigs immunized with ovine prolactin (PRL) were used in the development of a heterologous radioimmunoassay to measure PRL levels in sera and pituitary extracts in the white-tailed deer ( Odocoileus virginianus borealis). The RIA employs an antiserum with an association constant of 1.8 × 10 11 M −1 for 125IoPRL, which shows minimal (<0.2%) cross-reactivity with all other hypophyseal hormones tested (human PRL, GH, and CG; rat PRL and GH; bovine GH and TSH; ovine and equine LH; porcine ACTH and FSH), and has an ID 50 of 0.34 ± 0.02 ng against oPRL standards. Deer sera and hypophyseal extracts showed parallelism with standards. In adult males and females of a captive, but free-breeding population, baseline and thyrotropin releasing hormone (TRH)-stimulated (200 μg/deer, iv bolus) serum PRL values were measured. Baseline values for both sexes were similar and showed a seasonal variation with a summer peak of ∼ 150 ng/ml in June and winter levels of ∼ 1 ng/ml. Serum PRL concentrations were elevated by TRH injection in both sexes. Peak postinjection concentrations were attained within 20 min during April through September ( N = 49), or within 100 min in October through March ( N = 39). In contrast, LHRH (200 μg/deer, iv bolus) produced no significant change in PRL levels, while 2-bromo-α-ergocryptine (CB-154) reduced both baseline serum concentrations and TRH-induced elevations. Pituitary PRL levels in males and nonlactating females were similar and paralleled the serum PRL profile in the months tested, rising from about 1 μ/mg wet weight in winter to about 6μ/mg in summer.