Phthalate esters exert deleterious effects on testicular physiology and, consequently, on reproduction and fertility. However, little is presently known concerning potential adverse effects of these environmental pollutants on the hormonal functions of the adrenal gland. Therefore, we have investigated the effects of administering to rats of different developmental ages di-2-ethylhexyl phthalate (DEHP) on the hypothalamic–pituitary–adrenal axis in vivo, as well as on adrenocortical steroidogenesis ex vivo. Oral exposure to DEHP once daily for 4 days elevated the serum levels of ACTH and corticosterone in rats 20 and 40 days of age, but not in adult, 60-day-old animals. Furthermore, primary cultures of adrenocortical cells isolated from 20- and 40-day-old rats treated with DEHP exhibited an enhanced capacity to produce corticosterone in response to ACTH, dibutyryl cAMP, and 22R-hydroxycholesterol, as well as increased ACTH-stimulated transport of endogenous cholesterol into mitochondria. Neither DEHP nor its major metabolite mono-2-ethylhexyl phthalate altered steroidogenesis in cultures of adrenocortical cells isolated from untreated rats. These findings demonstrate that in male rats, DEHP exerts an age-dependent influence on the pituitary–adrenocortical axis in vivo and adrenocortical steroidogenesis ex vivo. Such perturbation may be of pathological significance in connection with disorders of the hormonal stress response, especially in very young human beings.