An antiserum to the opioid octapeptide met5-enk-arg6- gly7 -leu8 was used to measure the distribution and molecular weight heterogeneity of met5-enk-arg6- gly7 -leu8-immunoreactive (IR) peptides in rat brain. High concentrations of met5-enk-arg6- gly7 -leu8-IR peptides were found in the striatum and hypothalamus; low concentrations were observed in the cortex and cerebellum. Intermediate levels of immunoreactivity were found in other brain regions. Thus the distribution of met5-enk-arg6- gly7 -leu8-IR peptides closely parallels the distribution of met5-enkephalin. The immunoreactivity present in brain regions was characterized by gel filtration and reverse-phase high pressure liquid chromatography. In the striatum, more than 85% of the met5-enk-arg6- gly7 -leu8-IR peptides eluted from a Biogel P-30 column at the position of the authentic octapeptide; the identity of this material was further confirmed by HPLC. Gel filtration of extracts prepared from the medulla-pons resulted in two peaks of immunoreactive material of equal size; one eluted at approximately 8000 daltons and one eluted in the position of the octapeptide. Enzymatic digestion (trypsin + carboxypeptidase B) of the 8000 dalton-IR peptide resulted in the generation of met5-enkephalin-IR. Extracts prepared from other brain regions contained varying amounts of this high molecular weight form of met5-enk-arg6- gly7 -leu8.