High-affinity binding sites for the isoquinoline carboxamide PK 11195 and 4'-chlorodiazepam (4'CD) in human lymphocytes are recognized by two putative endogenous ligands: diazepam binding inhibitor (DBI) and protoporphyrin IX. We have now demonstrated that several synthetic DBI peptides--analogues to naturally processed human DBI (H-DBI) fragments--differ from protoporphyrin IX in the manner in which they displace [3H]PK 11195 and [3H]4'CD from binding sites associated with intact and cell-free lymphocyte preparations. In particular, the peptide fragments DBI37-80 and DBI37-70 displaced [3H]PK 11195 and [3H]4'CD with higher affinity from their binding sites on intact lymphocytes (Ki approximately 3-5 microM) than from the sites in the cell-free preparation (Ki approximately 20 microM). In contrast, protoporphyrin IX displaced [3H]PK 11195 and [3H]4'CD with higher affinity in the cell-free preparation (Ki - 0.4 microM) than in intact lymphocytes (Ki > 50 microM). Because DBI peptide fragments and protoporphyrin IX do not readily penetrate the plasma membrane of lymphocytes, our results suggest the existence of binding sites located both on the external face of the plasma membrane and intracellularly. The plasma membrane binding sites, recognized essentially only by DBI fragments, are termed here plasma membrane DBI receptors (PDRs). In contrast, the intracellular binding sites, recognized by both DBI fragments and protoporphyrin IX, are presumably located on mitochondria and are termed mitochondrial DBI receptors (MDRs). Immunohistochemical electronmicroscopic studies with antibodies to the synthetic peptide fragments 62-76 of the rat MDR support the hypothesis that PDRs are expressed on lymphocyte plasma membranes.