Abstract: The objective of this study was to apply the radioimmunoassay for 6-sulphatoxymelatonin (aMT6s) to rat urine, and use it to study the source of aMT6s. The radioimmunoassay was found to have acceptable within- and between-assay variation, excellent specificity, and good parallelism between the standard and unknown. Because urine is highly contaminated we assessed whether preliminary purification was required and established that it was unnecessary. Using this assay a 24-hr rhythm in 6-sulphatoxymelatonin output was seen in pools of urine harvested at 3-hr intervals from Wistar rats on LD 12:12. The nocturnal rise in aMT6s was abolished by constant light. In contrast pinealectomy lowered aMT6s output significantly throughout both dark and light. This study confirms previous studies indicating that the pineal is the major source of 6-sulphatoxymelatonin. It is concluded that urinary 6-sulphatoxymelatonin as measured by radioimmunoassay is a valid measure of pineal gland activity in the rat.