Purpose: The main goal of the present study was to investigate the importance of the addition of a positively charged aa in the naturally occurring Bombesin (BN) peptide for its utilization as radiodiagnostic agent, taking into consideration the biodistribution profile, the pharmacokinetic characteristics and the tumor targeting ability. Methods: Two BN-derivatives of the general structure [M-chelator]-(spacer)-BN(2–14)-NH2, where M: 99mTc or 185/187Re, chelator: Gly-Gly-Cys-, spacer: -(arginine)3-, M-BN-A; spacer: -(ornithine)3-, M-BN-O; have been prepared and evaluated as tumor imaging agents. Results: The peptides under study presented high radiolabelling efficiency (>98%), significant stability in human plasma (>60% intact radiolabelled peptide after 1 h incubation) and comparable receptor binding affinity with the standard [125I-Tyr4]-BN. Their internalization rates in the prostate cancer PC-3 cells differed, although the amount of internalized peptide was the same. The biodistribution and the dynamic -camera imaging studies in normal and PC-3 tumor-bearing SCID mice have shown significant tumor uptake, combined with fast blood clearance, through the urinary pathway. Conclusion: The addition of the charged aa spacer in the BN structure was advantageous for biodistribution, pharmacokinetics and tumor targeting ability, because it reduced the upper abdominal radioactivity levels and increased tumor/normal tissue contrast ratios.