The profiles of estradiol (E2), progesterone (P), and androstenedione (A), have been studied for the first time in cyclic Gentile di Puglia ewes. Estradiol peaks at estrus whereas progesterone levels are high during the luteal phase. Androstenedione does not show meaningful cyclic fluctuation. All hormones examined show also a pulsatile pattern when plasma was collected hourly.