Opioids, and numerous centrally active drugs, are metabolized by cytochrome P450 2D (CYP2D). There are sex and estrous cycle differences in brain oxycodone analgesia. Here we investigated the mechanism examining the selective role of CYP2D in the brain on sex, estrous cycle, and hormonal regulation. Propranolol, CYP2D-specific mechanism-based inhibitor, or vehicle was delivered into cerebral ventricles 24 hours before administering oxycodone (or oxymorphone, negative control) orally to male and female (in estrus and diestrus) rats. Ovariectomized and sham-operated females received no treatment, estradiol, progesterone or vehicle. Analgesia was measured using tail-flick latency, and brain drug and metabolite concentrations were measured by microdialysis. Data were analyzed by two-way or mixed ANOVA. Following propranolol (versus vehicle) inhibition and oral oxycodone, there were greater increases in brain oxycodone concentrations and analgesia, and greater decreases in brain oxymorphone/oxycodone ratios (an in vivo phenotype of CYP2D in brain) in males and females in estrus, compared to females in diestrus; with no impact on plasma drug concentrations. There was no impact of propranolol pre-treatment, sex, or cycle after oral oxymorphone (non-CYP2D substrate) on brain oxymorphone concentrations or analgesia. There was no impact of propranolol pre-treatment following ovariectomy on brain oxycodone concentrations or analgesia, which was restored in ovariectomized females following estradiol, but not progesterone, treatment. Sex, cycle, and estradiol regulation of CYP2D in brain in turn altered brain oxycodone concentration and response, which may contribute to the large inter-individual variation in response to the numerous centrally acting CYP2D substrate drugs, including opioids.