We have previously shown that oxidative stress within the tumor microenvironment causes phosphatidylserine (PS) to redistribute from the inner to the outer membrane leaflet of the endothelial cells (EC) creating a highly specific marker for the tumor vasculature. Because the distribution of phosphatidylethanolamine (PE) and PS within the membrane is coregulated, we reasoned that PE would also be localized in the outer membrane leaflet of tumor EC. To demonstrate this, the PE-binding peptide duramycin was biotinylated and used to determine the distribution of PE on EC in vitro and in vivo. Exposure of cultured EC to hypoxia, acidity, reactive oxygen species, or irradiation resulted in the formation of membrane blebs that were intensely PE-positive. When biotinylated duramycin was intravenously injected into tumor-bearing mice, it preferentially localized to the luminal surface of the vascular endothelium. Depending on tumor type, 13% to 56% of the tumor vessels stained positive for PE. PE-positive vessels were observed in and around hypoxic regions of the tumor. With the exception of intertubular vessels of the kidney, normal vessels remained unstained. To test the potential of PE as a biomarker for imaging, duramycin was conjugated to the near-infrared fluorophore 800CW and used for optical imaging of RM-9 prostate carcinomas. The near-infrared probe was easily detected within tumors in live animals. These results show that PE, like PS, becomes exposed on tumor vascular endothelium of multiple types of tumors and holds promise as a biomarker for noninvasive imaging and drug targeting.