Rat and guinea pig brain extracts were examined for the occurrence of gastrin-releasing peptide (GRP)-like substances by sequence specific radioimmunoassays interfaced with gel filtration and reversed phase high performance liquid chromatography (RP-HPLC). Tryptic digestion of the immunoreactive peptides followed by RP-HPLC was used to further characterize GRP-related peptides in brain. Using these analytical techniques it was found that guinea pig brain extracts contained a peptide with characteristics identical to authentic GRP (27 amino acid residues long). A carboxyterminal fragment with the characteristics of GRP(18-27) as well as a respective aminoterminal fragment with the characteristics of GRP(1-16) were also present in guinea pig brain extracts. The GRP(18-27) seems to correspond to the bombes in related material that has been described previously in mammalian brain extracts. Rat brain extracts also contained a peptide with the characteristics of GRP(18-27). The corresponding aminoterminal fragment, however, behaved differently on RP-HPLC from authentic GRP(1-16) and it was not recognized by antibodies directed to the aminoterminal tridecapeptide fragment of authentic GRP. Similarly the GRP-like peptide from rat brain did not comigrate on RP-HPLC with authentic GRP and was unreactive to antibodies directed toward the aminoterminus of GRP.