As a part of a study aiming to characterize the physiological and pharmacological significance of the high affinity pineal benzodiazepine (BZP) binding sites reported previously, we examined the uptake of the BZP derivative 3H-flunitrazepam (FNZP) by rat pineal glands in vitro. At 37 degrees C, 3H-radioactivity was taken up by tissue up to a pineal/medium concentration of about 12, while at 0 degrees C the uptake amounted to only one-third that at 37 degrees C. Reciprocal of uptake analyzed by Lineweaver-Burk plots indicated apparent Km's of 1.74 and 1.45 microM, and Vmax's of 1.32 and 1.04 pmol per min per mg tissue, for control and superior cervical ganglionectomized rats, respectively, suggesting that the neural compartment does not participate significantly in 3H-FNZP uptake. Cerebral cortex explants of similar size and weight as the pineal ones took up 3H-FNZP to a maximum tissue/medium concentration of about 2. Neither pineal nor cerebral cortex 3H-radioactivity uptake exhibited significant changes as a function of time of day. A number of agents, including several BZP analogues, cocaine, desipramine, melatonin, fluoxetine, nomifensine, and dipiridamol, as well as changes in the ionic environment or metabolic inhibitors, did not affect 3H-FNZP uptake significantly. Other tissues, such as liver, muscle, kidney, adrenal gland, or anterior pituitary, took up 3H-radioactivity to tissue concentrations slightly lower than those of the cerebral cortex, suggesting that drug liposolubility accounted only to a limited extent for the high in vitro uptake detected in incubated pineals.