In this study we examined the cerebrospinal fluid (CSF) for regional differences in the concentration of melatonin. Cerebrospinal fluid samples were collected at hourly intervals from either the lateral ventricle or the cisterna magna of nine Merino ewes and were compared against jugular plasma. The study revealed that the CSF of the cisterna magna and the lateral ventricle had temporal patterns of melatonin that were similar to those found in jugular plasma. However, the concentrations of melatonin within the CSF obtained from the lateral ventricle were one order of magnitude higher than those of the jugular plasma, as verified by gas chromatography and mass spectrometery, while the melatonin concentrations within the CSF obtained from the cisterna magna were comparable to those of the jugular plasma. The data from this study suggest that there may be regional differences in the concentration of melatonin within the CSF and indicate that this medium is an important route of transport for melatonin from the pineal gland to putative target tissues.