<jats:title>Abstract</jats:title><jats:p>Hydroxamamide (Ham) is a thiol-free chelating agent that forms technetium-99m (<jats:sup>99m</jats:sup>Tc)-complexes with a metal-to-ligand ratio of 1:2 under moderate reaction conditions. Therefore, Ham-based chelating agents will produce <jats:sup>99m</jats:sup>Tc-labeled compounds with a bivalent targeting scaffold. For their universal usage, we developed a novel Ham-based bifunctional chelating agent, “Ham-Mal”, with a maleimide group that can easily conjugate with a thiol group, for to preparing <jats:sup>99m</jats:sup>Tc-labeled bivalent ligand probes. Ham-Mal was synthesized by a four-step reaction, and then reacted with cysteine or c(RGDfC) to produce Ham-Cys or Ham-RGD. These precursors were reacted with <jats:sup>99m</jats:sup>TcO<jats:sub>4</jats:sub><jats:sup>-</jats:sup> for 10 min under room temperature to obtain <jats:sup>99m</jats:sup>Tc-(Ham-Cys)<jats:sub>2</jats:sub> and <jats:sup>99m</jats:sup>Tc -(Ham-RGD)<jats:sub>2</jats:sub>. The cellular uptake level of <jats:sup>99m</jats:sup>Tc-(Ham-RGD)<jats:sub>2</jats:sub> by U87MG (high Integrin ɑ<jats:sub>v</jats:sub>β<jats:sub>3</jats:sub> expression) cells was significantly higher than that by PC3 (low Integrin ɑ<jats:sub>v</jats:sub>β<jats:sub>3</jats:sub> expression) cells at 60 min after the incubation, and the uptake was significantly suppressed by pre-treatment for 15 min with excess c(RGDfK) peptide. In the in vivo study with U87MG/PC3 dual xenografted BALB/c-nu mice, the radioactivity of U87MG tumor tissue was significantly higher than that of PC3 tumor tissue at 360 min after the administration of <jats:sup>99m</jats:sup>Tc-(Ham-RGD)<jats:sub>2</jats:sub>. These results suggest Ham-Mal may have potential as a bifunctional chelating agent for <jats:sup>99m</jats:sup>Tc-labeled bivalent ligand probes. </jats:p>