Ethylene cross-bridged tetraamine macrocycles are useful chelators in coordination, catalytic, medicinal, and radiopharmaceutical chemistry. Springborg and co-workers developed trimethylene cross-bridged analogues, although their pendant-armed derivatives received little attention. We report here the synthesis of a bis-carboxymethyl pendant-armed cyclen with a trirnethylene cross-bridge (C3B-DO2A) and its isomeric ethylene-cross-bridged homocyclen ligand (CB-TR2A) as well as their copper(II) complexes. The in vitro and in vivo properties of these complexes are compared with respect to their potential application as Cu-64-radiopharmaceuticals in positron emission tomography (PET imaging). The inertness of Cu-C3B-DO2A to decomplexation is remarkable, exceeding that of Cu-CB-TE2A. Electrochemical reduction of Cu-CB-TR2A is quasi-reversible, whereas that of Cu-C3B-DO2A is irreversible. The reaction conditions for preparing Cu-64-C3B-DO2A (microwaving at high temperature) are relatively harsh compared to Cu-64-CB-TR2A (basic ethanol). The in vivo behavior of the Cu-64 complexes was evaluated in normal rats. Rapid and continual clearance of Cu-64-CB-TR2A through the blood, liver, and kidneys suggests relatively good in vivo stability, albeit inferior to Cu-64-CB-TE2A. Although Cu-64-C3B-DO2A clears continually, the initial uptake is high and only about half is excreted within 22 h, suggesting poor stability and transchelation of Cu to proteins in the blood and/or liver. These data suggest that in vitro inertness of a chelator complex may not always be a good indicator of in vivo stability.