Orexins are peptides controlling feeding, sleep and neuroendocrine functions. They are synthesized by the hypothalamus with projections throughout the brain. Orexins and their receptors OX₁ and OX₂ are present outside the CNS. Here the expression of preproorexin (PPO), OX₁ and OX₂ was studied in rat ovaries. PPO, OX₁ and OX₂ were determined by quantitative real-time RT-PCR in ovaries of cycling Sprague-Dawley rats on all days of the cycle. Serum hormones and food consumption were determined. Then, ovarian OX₁ and OX₂ expression was studied after ovulation blockade with Cetrorelix or Nembutal. Finally, proestrous rats were treated at 14:00 h and 19:00 h with a selective OX₁ antagonist (SB-334867-A) and/or a selective OX₂ antagonist (JNJ-10397049) and hormone levels, ovulation and ovarian histology were studied. Both receptors expression increased in the ovary between 17:00 h and 23:00 h of proestrus exclusively, in coincidence with hormone peaks, but not with the dark-light cycle or food intake. PPO was not detected. Cetrorelix or Nembutal prevented the increases of OX₁ and OX₂ while blunting gonadotropin peaks. SB-334867-A and JNJ-10397049, alone or combined, decreased serum gonadotropins and reduced ova number the following morning; ovaries showed a bloody (hyperemic and/or hemorrhagic) reaction with more preovulatory follicles and less corpora lutea. Here we demonstrate for the first time an increased ovarian expression of both OX₁ and OX₂, only during proestrous afternoon, and its hormone dependence but not on the dark-light cycle. Two new receptor antagonists reduced proestrous gonadotropins and/or ova number while producing ovarian structural changes.