Serum hormone levels of blood collected within 1 min by cardiac puncture following ether anesthesia were compared to those of blood collected by decapitation in male rats 30, 60, and 90 days of age. Neither testosterone (T) nor LH was affected by the method of blood collection. Serum FSH was significantly elevated in blood collected by cardiac puncture, and 30-day old rats were the most sensitive to this effect. Serum prolactin (Prl) was significantly elevated in all age groups when blood was collected by cardiac puncture. Both blood-collecting methods demonstrated that, in the 3 ages tested, serum T increased with age, LH and FSH decreased with age, and Prl did not change with age, indicating that blood collection by cardiac puncture is as valid a method as is decapitation for assessing serum hormone levels during maturation.