This report describes the cloning of a full length cDNA encoding PKBS, a protein of approximately 17 kDa associated with peripheral-type benzodiazepine binding sites. Cyanogen bromide digestion of purified PKBS yielded several peptide fragments which were subjected to gas-phase sequencing. Based on these partial amino acid sequences, oligonucleotide probes were used to screen a rat adrenal cDNA library. Several hybridizing clones were isolated which were found to contain overlapping sequences. The longest cDNA spanned 781 base pairs and specified an open reading frame of 169 amino acids which matched all of the peptide sequences. Northern analysis with this PKBS cDNA probe in different rat tissues revealed one RNA species of approximately 850 nucleotides exhibiting relative abundances qualitatively comparable with the densities of peripheral-type benzodiazepine binding sites in each tissue. The PKBS cDNA was cloned into an eukaryotic expression vector placing it under transcriptional control of the beta-globin promoter and SV40 enhancer. Transfection of the transformed human kidney 293 cell line with this recombinant vector resulted in stoichiometric increases of about 900 fmol/mg of protein in binding capacities for Ro5-4864 (4'-chlorodiazepam) and PK 11195, two peripheral-type benzodiazepine ligands. These increases were accounted for by the expression of binding sites with approximate dissociation constants of 5 nM for PK 11195 and 8 nM for Ro5-4864, thereby distinguishing the expressed binding sites as being characteristic of the receptor from rat origin rather than of the host human-derived cell line. The rank order of potency of different ligands to compete against [3H]Ro5-4864 binding in the transfected cells was PK 11195 greater than Ro5-4864 greater than diazepam greater than protoporphyrin IX greater than clonazepam, consistent with the specificity characteristic of rat peripheral-type benzodiazepine binding sites. These studies suggest that PKBS comprises binding domains for benzodiazepines and isoquinoline carboxamides and hence is apparently responsible for the manifestation of peripheral-type benzodiazepine recognition sites.