Purpose <p>Measuring hepatic flux rates of transportable substrates has the potential for assessing liver function. PET imaging of a PET-enabled substrate may provide a more straightforward measurement of time-dependent substrate concentration through the liver than MRI using an MRI contrast agent. Here we synthesized and evaluated the hepatobiliary transport of a new hepatospecific PET agent designed for stable Cu²⁺ chelation and transport by hepatic OATPs, [⁶⁴Cu]Cu-EOB-NOTA.</p> Procedures <p>EOB-NOTA was synthesized, its two enantiomers separated by chiral HPLC, and individually radiolabeled with [⁶⁴Cu]Cu²⁺. Cocktails of each enantiomer of [⁶⁴Cu]Cu-EOB-NOTA and Gd-EOB-DTPA were formulated for simultaneous PET/MRI imaging of hepatic flux by PET and MRI. Two mouse models were evaluated: wild-type mice and mice expressing only human hepatic OATPs.</p> Results <p>In wild-type mice, [⁶⁴Cu]Cu-EOB-NOTA hepatic influx and efflux was high, but slower compared to Gd-EOB-DTPA. Neither enantiomer of [⁶⁴Cu]Cu-EOB-NOTA exhibited detectable transport into the liver in mice expressing human OATPs. This was validated by waste clearance studies and <i>in vitro</i> uptake assays in cells engineered to express rodent and human OATPs.</p> Conclusion <p>[⁶⁴Cu]Cu-EOB-NOTA exhibited no detectable hepatic uptake by transgenic mice expressing human hepatic transporters. This finding was surprising given the efficient transport of the structurally similar metal chelate Gd-EOB-DTPA, and underscores challenges in the&#xa0;design of imaging molecular probes, including poor predictability for hepatic transport, and the value of validating new agents in mice expressing human hepatic transporters.</p>