In 33 normal infants, divided into 3 age groups (less than 1 month, 1-3 months, and 3-6 months) plasma cortisol was measured at 2230, 2300, and 2330 h. Baseline plasma cortisol at 2230 h. was, as expected, low in all infants, with mean +/- SEM values of 41 +/- 5, 72 +/- 14, and 97 +/- 17 nmol/L in each group, respectively. Thirty and 60 min after the painful stimulus of the venipuncture, plasma cortisol increased significantly (P less than 0.0005), reaching a maximum increase up to 458 +/- 50, 392 +/- 66, and 455 +/- 97 nmol/L in each age group, respectively. We conclude that in these infants the hypothalamic-pituitary-adrenal axis was functionally intact and responded to the painful stimulus of the venipuncture by a significant increase in plasma cortisol. This test may be used as a simple procedure for the evaluation of the integrity of the hypothalamic-pituitary-adrenal axis without the administration of pharmacological agents. Its usefulness, however, should be validated with patients having a disorder of the system.