A sensitive, specific radioimmunoassay for bovine α-lactalbumin (α-la) suitable for measurement of serum, tissue culture media and milk was developed. α-La was not detected in serum from prepubertal male or female cattle, but was detected as early as 60 d of gestation in nulliparous Holstein heifers, the level being greatly increased during the last one third of gestation. Serum from cross bred beef heifers contained less α-la and it was not detected until late gestation. Concentrations of α-la in serum samples from pregnant multiparous Holstein cows decreased at drying-off and subsequently increased just before parturition. Secretion of α-la by mammary tissue explants from steroid-primed prepubertal Holstein heifers was induced by the addition of bovine prolactin, ovine prolactin or human growth hormone to tissue culture media.