Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae and extracts prepared from rat adrenal gland and striatum were subjected to Sephadex G-50 chromatography and HPLC. Fractions were monitored using specific radioimmunoassays (RIA) for the pentapeptide methionine enkephalin (Met-Enk) and methionine enkephalin-Arg6-Gly7-Leu8 (Met-EnkRGL). In rat CSF, striatum and adrenal gland, three Met-EnkRGL-immunoreactive (IR) peaks of Mrs 8000, 5000 and 1000 daltons were detected. The same peaks were also found to possess Met-Enk-immunoreactivity after enzyme digestion of Sephadex G-50 fractions with trypsin and carboxypeptidase B (CPB), suggesting their derivation from proenkephalin. HPLC of the 8K and 5K peaks on a column of Ultrapore RPSC showed them to elute discretely with similar retention times, indicative of hydrophobic peptides of large molecular weight. Their similar hydrophobicities yet significant separation during gel filtration would suggest that the 8K and 5K peptides are structurally closely related yet different with respect to their molecular weights. HPLC of the small molecular weight material from rat striatum and adrenal gland revealed the presence of Met-EnkRGL and Met-EnkRGL sulphoxide in both tissues. In rat striatum Met-Enk and its sulphoxide were also detected. The oxidised pentapeptide was found to be present in rat CSF, together with two unidentified small molecular weight Met-Enk-IR peaks detected without prior enzyme digestion of fractions. The small molecular weight Met-EnkRGL-IR material in rat CSF was found to be comprised of two unknown peptides which were less hydrophobic than Met-EnkRGL and its sulphoxide derivative.