Molecular imaging can report on the status of the tumor immune microenvironment and guide immunotherapeutic strategies to enhance the efficacy of immune modulation therapies. Imaging agents that can rapidly report on targets of immunomodulatory therapies are few. The programmed death ligand 1 (PD-L1) is an immune checkpoint protein over-expressed in several cancers and contributes to tumor immune suppression. Tumor PD-L1 expression is indicative of tumor response to PD-1 and PD-L1 targeted therapies. Herein, we report a highly specific peptide-based positron emission tomography (PET) imaging agent for PD-L1. We assessed the binding modes of the peptide WL12 to PD-L1 by docking studies, developed a copper-64 labeled WL12 ([Cu-64] WL12), and performed its evaluation in vitro, and in vivo by PET imaging, biodistribution and blocking studies. Our results show that [Cu-64] WL12 can be used to detect tumor PD-L1 expression specifically and soon after injection of the radiotracer, to fit within the standard clinical workflow of imaging within 60 min of administration. (C) 2016 Elsevier Inc. All rights reserved.