Diazepam binding inhibitor (DBI), a 11,000 MW neuropeptide, which coexists with GABA and elicits proconflict responses in the rat, has been purified and partially sequenced from rat brain. We now report purification and characterization of a DBI-like neuropeptide from human brain. Its molecular weight and pharmacological profile is identical to that of rat DBI but differs in the amino acid composition and immunologically. The tryptic fragments of human DBI differ from rat DBI in the HPLC elution profile and in the amino acid sequence. Using high affinity specific human DBI antibodies, the distribution of DBI-like immunoreactivity in bioptic samples of human brain appeared to be similar to that of DBI found in rat brain. DBI-like immunoreactivity was also found in spinal fluid of human volunteers. The cerebrospinal fluid content of this peptide might be used as a probe to study whether spinal fluid DBI content changes in neuropsychiatric disorders.