Milk was collected for the first 21 days of lactation twice daily from dairy cows and once daily from goats, sheep, and guinea pigs. Thyroxine (T4) and triiodothyronine (T3) were extracted from 100 microliter of milk using acidified ethanol. T4 and T3 were reconstituted in 100 microliter buffer and measured by radioimmunoassay. Concentrations (ng/ml) of T4 and T3 for milk of cows, goats, sheep, and guinea pigs, respectively, were: 0.97 and 0.94, 1.24 and 0.52, 0.99 and 0.79, and 1.41 and 0.53. T4 concentration for guinea pig milk was significantly higher than for cow and sheep milk, but not for goat milk (P less than 0.05). T3 was found in higher concentration in milk of cows and sheep than in milk of goats and guinea pigs (P less than 0.05). Species differences in conversion of T4 to T3 in mammary gland cells are suggested. Summations of T4 and T3 concentrations in milk indicated no differences among the four species. Regression analyses of changes in milk production, T4 and T3 concentrations, total T4 and T3 in milk per day, and ratios of T4 to T3 revealed variations in patterns. Concentrations of T4 or T3 tended to decrease as lactation progressed over 21 days. Total T3 tended to increase, and the ratio of T4 to T3 tended to decrease. Amounts of T4 and T3 available to offspring from milk were calculated to be minor sources (4 to 7%) of total requirements for maintenance of metabolic functions.