Programmed death ligand 1 (PD-L1)–mediated immunosuppression regulates peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but also binds to B7-1 (CD80) to regulate T-cell function. The binding interaction of PD-L1 with B7-1 and its functional role need further investigation to understand differences between PD-1 and PD-L1 tumor immunotherapy. We examined the molecular orientation of PD-L1 binding to B7-1 using cell-to-cell binding assays, ELISA, and flow cytometry. As expected, PD-L1–transfected cells bound to PD-1–transfected cells, and B7-1 cells bound to CD28 or CTLA-4–transfected cells; however, PD-L1 cells did not bind to B7-1 cells. By ELISA and flow cytometry with purified proteins, we found PD-L1 and B7-1 had a strong binding interaction only when PD-L1 was flexible. Soluble PD-1 and B7-1 competed for binding to PD-L1. Binding of native PD-L1 and B7-1 in cis on the same cell surface was demonstrated with NanoBiT proximity assays. Thus, PD-L1–B7-1 interaction can occur in cis on the same cell but not in trans between two cells, which suggests a model in which PD-L1 can bend via its 11-amino acid, flexible stalk to bind to B7-1 in cis, in a manner that can competitively block the binding of PD-L1 to PD-1 or of B7-1 to CD28. This binding orientation emphasizes the functional importance of coexpression of PD-L1 and B7-1 on the same cell. We found such coexpression on tumor-infiltrating myeloid cells. Our findings may help better utilize these pathways in cancer immunotherapy. Cancer Immunol Res; 6(8); 921–9. ©2018 AACR.