In this paper the preclinical evaluation of the somatostatin analogue RC160 labelled with technetium-99m using bifunctional chelators (BFCs) based on the hydrazinonicotinamide (HYNIC) and N(3)S system is described and a comparison made with [Tyr(3)]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG(3) and an N(3)S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands for HYNIC conjugates. All conjugates and (99m)Tc-labelled peptides showed preserved binding affinity for the somatostatin receptor (IC50, Kd<5 nM). The biodistribution was markedly dependent on the BFC and co-ligand used, with the amidothiol ligands showing a greater degree of hepatobiliary clearance, the HYNIC/tricine complex higher blood levels and the HYNIC/EDDA complex the highest level of renal excretion and lowest blood levels. All peptide conjugates showed receptor-mediated uptake in tumour xenografts, but tumour uptake was significantly lower for the (99m)Tc-RC160 derivatives compared with (99m)Tc-EDDA/HYNIC-[Tyr(3)]-octreotide (0.2%-3.5%ID/g vs 9.7%ID/g) and correlated well with the reduced internalisation rate for RC160 derivatives. Our results show that the selection of the labelling approach as well as the right choice of the peptide structure are crucial for labelling peptides with (99m)Tc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with (99m)Tc-EDDA/HYNIC-[Tyr(3)]-octreotide, (99m)Tc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives.