DRAP27, the monkey homolog of human CD9 antigen (DRAP27/CD9) and diphtheria toxin receptor (DTR) were expressed in mouse L cells. L cells transfected transiently with both DRAP27/CD9 and DTR cDNA bound approximately 10 times more diphtheria toxin (DT) than cells transfected with DTR alone. Stable L cell transfectants expressing both DTR and DRAP27/CD9 (LCH-1 cells) had 15 times more cell surface DT-binding sites and were 20 times more sensitive to DT than were stable L cell transfectants expressing DTR alone (LH-1 cells). Increased DT-binding and DT sensitivity were not due to increased DTR transcription or increased cell surface DTR protein. Co-immunoprecipitation of DRAP27/CD9 with DTR and chemical cross-linking suggest a tight association of these membrane-bound proteins. In addition, the identity of DTR and a growth factor (HB-EGF) was established. Immobilized DT specifically adsorbed HB-EGF precursor solubilized from transfected L cells and [125I]DT bound to immobilized recombinant HB-EGF. We conclude that DRAP27/CD9 associates tightly with DTR/HB-EGF and up-regulates the number of functional DTRs and DT sensitivity, and that HB-EGF is identical to DTR.