Analogues of the macrocyclic chelator 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) are currently used as chelators of choice for labeling copper radionuclides to biological molecules. Recently, the "cross-bridged" cyclam complex, 64 Cu(II)-4,11bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) was shown to have improved in vivo stability compared to 64 Cu(II)-TETA (1). We hypothesized that a bifunctional chelator of CB-TE2A would be a good candidate for radiolabeling copper to biological molecules for imaging and radiotherapy. However, it was postulated that both -COOH groups are required to stably bind Cu(II) to form a neutral, hexacoordinate complex; when CB-TE2A is conjugated to a peptide, one carboxylate is conve rted to an amide linkage. To evaluate our hypothesis, 64 Cu-CB-TE2A-Y3-octreotate ( 64 Cu-CB-TE2A-Y3-TATE) was synthesized and evaluated in vivo in a tumorbearing rat model. Methods: CB-TE2A-Y3-TATE was synthesized by solid-phase methods using standard Fmoc chemistry and radiolabeled with 64 Cu in greater than 95% purity. Biodistribution and microPET (Concord Microsystems) experiments were performed in AR42J rat pancreatic tumorbearing male Lewis rats. Results: Initial microPET imaging of 64 Cu-CB-TE2A-Y3-TATE in AR42J bearing rats showed the highest non-target uptake in the kidneys (1.76 + 0.04 %ID/g at 1 h), with significant clearance by 24 h post-injection (0.32 + 0.03 %ID/g). Liver uptake was 0.31 + 0.02 %ID/g at 1 h and decreased to 0.051 + 0.002 %ID/g by 24 h post-injection demonstrating that the primary mode of clearance is renal rather than hepatobiliary. Quantification of activity in rat tumor, liver, and kidney was similar to in vivo biodistribution results. In biodistribution experiments, somatostatin-receptor positive tissues displayed significantly higher uptake at all time points compared to non-target organs; this was blocked by co-injection of 150 mg Y3-TATE (pancreas: 1 h = 6 + 1 %ID/g, 1h block = 0.37 + 0.03 %ID/g; AR42J tumor 1 h = 2.4 + 0.4 %ID/g, 1 h block = 0.8 + 0.1%ID/g). Blood clearance of 64 Cu-CB-TE2A-Y3-TATE compared to 64 Cu-TETA-Y3-TATE was found to be much more favorable. From 1 to 24 h post-injection, levels of 64 Cu-CB-TE2A-Y3-TATE in the blood decreased from 0.13 + 0.01 to 0.014 + 0.001% ID/g, while there was essentially no blood clearance of 64 Cu-TETA-Y3-TATE (0.09 + 0.01 to 0.10 + 0.01% ID/g) (2). This strongly suggests enhanced in vivo stability of the 64 Cu-CB-TE2A-Y3-TATE complex compared to 64 Cu-TETA-Y3-TATE. The enhanced blood clearance of 64 Cu-CB-TE2A-Y3-TATE gives rise to impressive tumor:blood ratios (156:1) at 4 h post-injection. Conclusions: These data indicate that CB-TE2A is a significantly improved bifunctional chelator compared to TETA for labeling 64 Cu to biological molecules such as the somatostatin analogue Y3-TATE and that only one -COOH group on CB-TE2A is required to bind Cu(II). Acknowledgements: The authors would like to thank Lynne Jones for technical assistance and acknowledge funding by NCI grant (CA93375).