Two somatostatin analogues, [(99m)Tc]Demotide and [(99m)Tc]Demotate 4, were compared with [(99m)Tc]Demotate 1, a previously reported somatostatin receptor subtype 2 (sst(2)) targeting tracer. Conjugates were prepared by coupling an open-chain tetraamine chelator to D-Phe(1) of [Tyr(3)]-octreotide or [Tyr(3)]-octreotate, respectively, via a p-benzylaminodiglycolic acid spacer adopting solid-phase peptide synthesis techniques. Peptide conjugates were collected in a highly pure form after chromatographic purification. Eventually, [(99m)Tc]Demotide and [(99m)Tc]Demotate 4 were obtained in approximately 1 Ci/micromol specific activity and >96% purity after labeling under alkaline conditions. Demotide and Demotate 4 exhibited similar high binding affinities for the sst(2) expressed in AR4-2J cells with IC(50) values 0.16 and 0.10 nM, respectively. The (radio)metallated analogues [(99m)Tc]Demotide and [(99m)Tc]Demotate 4 showed equally high affinities to the sst(2) during saturation binding assays in AR4-2J cell membranes (K(d)s 0.08 and 0.07 nM, respectively). During incubation at 37 degrees C with AR4-2J cells, the radiopeptides internalized effectively via a receptor-mediated process, with [(99m)Tc]Demotate 4 exhibiting a faster internalization rate than [(99m)Tc]Demotide. After injection in athymic mice bearing sst(2)-expressing AR4-2J tumors, the radiotracers showed high and specific uptake in the tumor (>25%ID/g at 1 h) and in the sst(2)-positive organs. However, both [(99m)Tc]Demotide and [(99m)Tc]Demotate 4 showed unfavorably higher background activity, especially in the abdomen, in comparison to [(99m)Tc]Demotate 1 and are, therefore, less suited than [(99m)Tc]Demotate 1 for sst(2)-targeted tumor imaging in man.