Fluorescein was found to penetrate into the brain via the circumventricular organs. Fluorescein penetrates beyond the borders of the circumventricular organs into the surrounding neuropile. This relationship was found for the area postrema and the nucleus tractus solitarius, the organum vasculosum lamina terminalis and the rostral suprachiasmatic area, the median eminence and the arcuate nucleus. The choroid plexus appears to take up fluorescein, but fluorescence does not appear in adjacent structures such as the corpus striatum and septum, but rather along the ependymal and plial surfaces. Fluorescein was found to accumulate to a greater extent in the brains of female as compared to male rats. This sex difference is not associated with the blood-brain barrier as cortex and cerebellum did not show increased fluorescence, but only midline structures containing circumventricular organs. Gonadectomy did not alter fluorescein accumulation.