Human cerebrospinal fluid was chromatographed on Bio-Gel P-4. Fractions containing material with molecular weights less than 4000 Dalton were pooled and further fractionated by high pressure liquid chromatography on an UltroPack TSK column G 2000 SW. At least three peaks, which were free of salt and GABA, were shown to displace (3H)-diazepam in the receptor-binding assay. Two of these peaks inhibited diazepam-binding competitively as shown by Lineweaver-Burke and displacement analysis. Their activity could be enhanced by the addition of GABA to the assay mixture. Incubation of these two peaks with various enzymes indicated that at least part of the activity of the second peak is due to a peptide.