Aromatase P450 (P450 AROM), converting testosterone (T) into estradiol (E), plays an important role in sexual differentiation of neural structures in the developing mammalian brain. The aim of the present study was to characterize the qualitative and quantitative profile of P450 AROM mRNA expression in the bovine hypothalamus (the region of the central nervous system in which the enzyme is mainly localized) using RT-PCR and quantitative real-time RT-PCR analysis, respectively. P450 AROM expression was examined in the developing hypothalamus in a series of experimental groups investigated at 10 weeks interval one from the other. Our data indicate that in the bovine fetal hypothalamus P450 AROM expression peaks at the second quarter of gestation. The presence of neural cells containing P450 AROM in the bovine fetal hypothalamus was confirmed by immunohistochemistry, and localized in the medial preoptic area. We conclude that second quarter of the gestation is the developmental stage which represents a critical period for hypothalamic differentiation in bovine ontogenesis, an important difference with the rat and mouse, short gestation species in which P450 AROM activity peaks around delivery.