A recent survey found that approximately 4% of very low birth weight infants in Japan were treated with glucocorticoids postnatally for circulatory collapse thought to be caused by late-onset adrenal insufficiency. We identified 11 preterm infants with clinical signs compatible with this diagnosis (hypotension, oliguria, hyponatremia, lung edema, and increased demand for oxygen treatment) and matched them for gestational age with 11 infants without such signs. Blood samples were obtained for cortisol and its precursors from the patient group before the administration of hydrocortisone, and from the control group during the same postnatal week. All samples were analyzed using a gas chromatography-mass spectrometry system. Cortisol concentrations did not differ between the two groups (6.6 ± 4.5 vs 3.4 ± 2.7 μg/dL); however, the total concentration of precursors in the pathway to cortisol production was significantly higher in the patient group (72.2 ± 50.3 vs 25.0 ± 28.5 μg/dL; p < 0.05). We conclude that the clinical picture of late-onset adrenal insufficiency in preterm infants is not a result of an absolute deficiency of cortisol production, but may be a result of a limited ability to synthesize sufficient cortisol for the degree of clinical stress.