High‐pressure liquid chromatography with electrochemical detection has been used for the determination of melatonin in rat pineal glands and tissue explants. Rat pineal melatonin content was measured at 2‐hr intervals during a 2.t‐hr period by direct injection of supernatants from centrifuged pineal homogenates into a reversed‐phase C18 column. The lower limit of sensitivity was 32 pg per gland for a 2:1 signal‐to‐noise ratio, which allows melatonin measurements during daytime. Both absolute values and circadian rhythmicity obtained with the present method are in agreement with previously validated rddioimmunoassays. In addition, isoproterenolinduced release of melatonin from individual rat pineal explants was measured after chloroform extraction of the incuba