Purpose: We aimed at designing and developing a novel bombesin analogue, DOTA-PEG4-BN(7-14) (DOTA-PESIN), with the goal of labelling it with 67/68Ga and 177Lu for diagnosis and radionuclide therapy of prostate and other human cancers overexpressing bombesin receptors. Methods: The 8-amino acid peptide bombesin (7-14) was coupled to the macrocyclic chelator DOTA via the spacer 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4). The conjugate was complexed with Ga(III) and Lu(III) salts. The GRP receptor affinity and the bombesin receptor subtype profile were determined in human tumour specimens expressing the three bombesin receptor subtypes. Internalisation and efflux studies were performed with the human GRP receptor cell line PC-3. Xenografted nude mice were used for biodistribution. Results: [GaIII/LuIII]-DOTA-PESIN showed good affinity to GRP and neuromedin B receptors but no affinity to BB3. [67Ga/177Lu]-DOTA-PESIN internalised rapidly into PC-3 cells whereas the efflux from PC-3 cells was relatively slow. In vivo experiments showed a high and specific tumour uptake and good retention of [67Ga/177Lu]-DOTA-PESIN. [67Ga/177Lu]-DOTA-PESIN highly accumulated in GRP receptor-expressing mouse pancreas. The uptake specificity was demonstrated by blocking tumour uptake and pancreas uptake. Fast clearance was found from blood and all non-target organs except the kidneys. High tumour-to-normal tissue ratios were achieved, which increased with time. PET imaging with [68Ga]-DOTA-PESIN was successful in visualising the tumour at 1h post injection. Planar scintigraphic imaging showed that the 177Lu-labelled peptide remained in the tumour even 3days post injection. Conclusion: The newly designed ligands have high potential with regard to PET and SPECT imaging with 68/67Ga and targeted radionuclide therapy with 177L