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The uptake of estradiol and progesterone into the cerebrospinal fluid (CSF) after intranasal and intravenous administration in rats was investigated. Each animal received estradiol intranasally (40 μg/rat) and by intravenous infusion (10 μg/rat) into the jugular vein using a vascular access port. Hereafter, the same set of rats was treated with progesterone intranasally (200 μg/rat) and by intravenous infusion (104 μg/rat). Following nasal delivery, both steroid hormones reach Cmax values in plasma and CSF at 15 min after administration. Intravenous infusion of estradiol and progesterone shows comparable plasma and CSF concentration–time profiles compared to the nasal route. For both hormones the AUCCSF/AUCplasma ratios (mean±SD) after intranasal delivery (estradiol 2.3±1.1%; progesterone 1.9±0.7%) do not differ significantly from the ratios shown after intravenous infusion (estradiol 2.0±0.6%; progesterone 2.2±0.8%). These results indicate that after nasal delivery estradiol and progesterone are rapidly absorbed into the systemic circulation, from where the non-protein bound hormones probably enter the CSF by crossing the blood–brain barrier. No extra direct nose–CSF transport could be demonstrated.