The diphtheria toxin receptor (DTR) has been identified as the precursor of heparin-binding epidermal growth factor-like growth factor, which may interact with other membrane proteins to form the functional receptor. To test if mammalian DTR is able to confer toxin sensitivity onto phylogenetically distant cells, we expressed monkey DTR in the baculovirus system and tested infected insect cells for toxin sensitivity. cDNA encoding an epitope-tagged heparin-binding epidermal growth factor-like growth factor precursor (DTRB3) was inserted into the virus genome by allelic replacement to construct the recombinant virus vAc-DTRB3. SF9 cells infected with vAc-DTRB3 expressed functional DTR, which could be precipitated from the solubilized membrane fraction of infected cells with Sepharose-immobilized diphtheria toxin. The highest level of expression (about 5 x 10(6) receptors/cell) was observed 48 h after infection, at which time the infected cells were highly sensitive to diphtheria toxin. Uninfected SF9 cells and cells infected with the wild type virus were resistant to the toxin. The presence of heparin increased both the binding and the toxin sensitivity of vAc-DTRB3-infected SF9 cells. Translocation of toxin A fragment was induced when cells with surface-bound toxin were exposed to low pH, and the translocation was optimal at pH < or = 5.5. It was approximately 100 times more efficient at 24 degrees C than at 4 degrees C. The data indicate that monkey DTR is fully functional when expressed in insect cells.