Abstract The development of a PET/optical imaging system is attractive because of the opportunity to obtain complementary data from two similar contrast mechanisms using one probe. Different from PET/CT or SPECT/CT, a linker is generally needed to efficiently incorporate PET tag, optical tag, and the biomarker together. Previously we developed a dicarboxyl-functionalized chelator (BaBaSar) derivatized from sarcophagine cage (denoted as Sar). BaBaSar has been used for 64Cu radiopharmaceutical development and the great in vivo stability of 64Cu-BaBaSar encouraged us to continue to derivatize the sarcophagine core for versatile conjugation styles with biomarkers. In this study, we synthesized a hetero-functionalized sarcophagine named BaAnSar: carboxyl group at one end of the Sar cage and amino group on the other end. The pendant hetero-arm could be further conjugated to multiple targeting ligands via biologically stable amide bonds. In order to prove the advantage of the this BaAnSar chelator, we chose dimeric c(RGDyK) peptide (denoted as RGD2), a well-known ligand targeting integrin αvβ3, onto the carboxyl side and fluorescent dye Cy5.5 on the amino functional side of the Sar cage. After labeling with 64Cu, a dual-modality probe, 64Cu-BaAnSar-RGD2-Cy5.5 was made and evaluated in U87MG tumor bearing nude mice. Both NIR fluorescence imaging and microPET clearly delineated the U87MG tumor from the surrounded normal tissue at 1, 4, and 20 h post injection of 64Cu-BaAnSar-RGD2-Cy5.5. The microPET quantification of U87MG tumor uptake was determined as 6.41 ± 0.28, 6.51 ± 1.45, and 5.92 ± 1.57 %ID/g at 1, 4, and 20 h post injection, respectively. Good correlation was also observed between the results measured by ex vivo PET and NIRF organ imaging. In conclusion, we have successfully developed a bifunctional chelator for dual PET and NIRF imaging probe construction. The BaAnSar chelator developed herein has the potential to enhance resolution for comprehensive visualization of the probe distribution and provides a rapid and efficient imaging method for basic research and potential clinical applications, especially as a visual guide during surgery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5731. doi:1538-7445.AM2012-5731