Abstract Photothermal ablation (PTA) is an emerging technique that uses near-infrared (NIR) laser light-generated heat to destroy tumor cells. Doxorubicin (DOX)-loaded hollow gold nanospheres (DOX@HAuNS) mediated simultaneous PTA of cancer cells and local drug release, resulting in enhanced antitumor activity. EphB4, a member of the largest family of receptor tyrosine kinases, is overexpressed in numerous tumors. The purpose of this study was to synthesize DOX@HAuNS conjugated to an EphB4 receptor-binding peptide, and evaluate the pharmacokinetics, biodistribution, and antitumor efficacy of the resulting EphB4-targeting DOX@HAuNS. A cyclic 17-mer peptide [c(TNYL-RAW) with high binding affinity to EphB4 was conjugated to DOX@HAuNS through a PEG linker. The cell uptake of c(TNYL-RAW)-conjugated Dox@HAuNS was examined in human ovarian cancer Hey cells over-expressing EphB4. The biodistribution tumor uptake of c(TNYL-RAW)-Dox@HAuNS was assessed at 24 h after intravenous injection in nude mice bearing Hey and A2789 ovarian cancers, and in nude mice bearing human MDA-MB231 breast tumors. Near-infrared (NIR) light-triggered Dox release in tumor-bearing mice was evaluated using a dual tracer-autoradiography technique. Antitumor effect was evaluated by measuring the tumor grow delay in mice bearing Hey ovarian cancer. In vitro, c(TNYL-RAW)-Dox@HAuNS were selectively taken up by Hey cells. This resulted in increased tumor uptake of targeted nanoparticles in all three EphB4-positive tumor models evaluated. Significantly higher cell killing effect against EphB4(+) A2780 cells was shown with DOX@c(TNYL-RAW)-HAuNS cells than with free DOX or DOX@PEG-HAuNS. In vivo, conjugation of c(TNYL-RAW) onto PEG-HAuNS did not cause significant change on pharmacokinetic properties of the nanoaprticles. DOX@c(TNYL-RAW)-HAuNS and DOX@PEG-HAuNS were almost completely eliminated from the blood at 48 h after injection. Treatment with targeted c(TNYL-RAW)-Dox@HAuNS followed by near-infrared laser irradiation (combined PTA and DOX therapies) resulted in significantly increased tumor growth control as compared to treatments with DOX@PEG-HAuNS-plus-laser (combined PTA and DOX therapies) and with PEG-HAuNS-plus-laser (PTA therapy alone). Mice in all treatment groups did not show lost in body weight, whereas free DOX caused ∼20% body weight loss at the equivalent DOX dose level. Targeted photothermal-chemotherapy exemplified by a single-agent nanoconstruct c(TNYL-RAW)-DOX@HAuNS is a promising approach to effective anticancer therapy. 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 5694. doi:1538-7445.AM2012-5694