Thyroid hormones (TH) in milk can be measured by RIA after prolonged extraction with alkaline ethanol at low temperature. The results of this method agreed with those obtained by gas chromatography-mass spectrometry, though they show the concentrations lower than in most of the already published studies based on radioimmunoassay. The levels of thyroxine (T4) in colostrum and milk in rabbit, cow and women were found to be similar, about 2 nmol l-1, which represented a small fraction of those in blood serum. Triiodothyronine (T3) content approximated one third of that in serum and was about 1.0, 1.8, 0.3 and 0.5 nmol l-1 in the rabbit, pig, human and cow, respectively. Although in a relatively high blood concentration, reverse-T3 does not appear in cow milk, but it passes through the blood/mammary gland barrier into the milk in rabbits in which it appears in a very low concentration. Of TH in milk, T3 may exert some physiological role in offsprings, particularly during the early adaptive postnatal period. Insulin (INS) in human, cow and pig milk was found in concentrations approximate to those in blood serum. They were high at the peri-parturient period and then fell rapidly in the pig and more gradually in the women, to a relatively low and constant levels. Transfer of INS into the mammary gland and milk was positively related to the actual concentrations of this hormone in the blood, in quantities which, following ingestion of milk, might exert some biological action in neonates.