To assess the effect of breast feeding on circulating thyroid hormone levels in the suckling neonate, we examined serum concentrations of T4, T3, and rT3 by RIA in 12- and 15-day-old rat pups fasted for 3 or 6 h before, at, and hourly for 3 or 4 h after suckling. In both age groups, serum T4 levels decreased significantly in response to the 6-h fast; no significant differences were observed after 3 h. T3 levels in 15-day-old (but not 12-day-old) rats were likewise significantly depressed, and rT3 levels were higher relative to those in the prefast group. After a 90-min suckling period, serum total and free T4 concentrations of 12-day-old pups increased 1 h postsuckling to levels significantly higher than those in the fasted animals; maximum levels were achieved at 2 h. T3 levels were unchanged over this period. In contrast, elevated total and free T4 and T3 levels and a reciprocal decrease in rT3 concentration, were evident immediately after the 90-min suckling period (0 h) in 15-day-old rat pups and increased through 1 h to levels significantly higher than those in the prefast group. After refeeding (0 h) of 3-h fasted pups, serum T4 levels increased significantly over fasted levels in suckled 12- and 15-day-old rats, as did T3 levels in 15-day-old pups, through 2 h postsuckling. In contrast, thyroid hormone levels of pups gavage-fed a commercial formula preparation at no time exceeded those of the prefast groups at either age, despite highly comparable stomach weights for both formula-fed and suckled pups at the time of refeeding. We conclude that 1) rat pups between the ages of 12 and 15 days postpartum become hypothyroxinemic between 3 and 6 h of milk deprivation, and 2) this condition is reversed by significant increases in T4 and T3, rendering the animal transiently hyperthyroxinemic after suckling, but not in response to formula feeding. Our data further demonstrate the critical nature of study designs involving fasts (either planned or incidental) of suckling rats in light of the observed pronounced variations in their thyroid hormone status relative to time postsuckling.