The overexpression of gastrin-releasing peptide receptor (GRPR) in cancer can be used for peptide-receptor mediated radionuclide imaging and therapy. We have previously shown that an antagonist analog of bombesin RM26 conjugated to 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA) via a diethyleneglycol (PEG2) spacer (NOTA-PEG2-RM26) and labeled with 68Ga can be used for imaging of GRPR-expressing tumors. In this study, we evaluated if a variation of mini-PEG spacer length can be used for optimization of targeting properties of the NOTA-conjugated RM26. A series of analogs with different PEG-length (n = 2, 3, 4, 6) was synthesized, radiolabeled and evaluated in vitro and in vivo. The IC50 values of natGa-NOTA-PEGn-RM26 (n = 2, 3, 4, 6) were 3.1 ± 0.2, 3.9 ± 0.3, 5.4 ± 0.4 and 5.8 ± 0.3 nM, respectively. In normal mice all conjugates demonstrated similar biodistribution pattern, however 68Ga-NOTA-PEG3-RM26 showed lower liver uptake. Biodistribution of 68Ga-NOTA-PEG3-RM26 was evaluated in nude mice bearing PC-3 (prostate cancer) and BT-474 (breast cancer) xenografts. High uptake in tumors (4.6 ± 0.6%ID/g and 2.8 ± 0.4%ID/g for PC-3 and BT-474 xenografts, respectively) and high tumor-to-background ratios (tumor/blood of 44 ± 12 and 42 ± 5 for PC-3 and BT-474 xenografts, respectively) were found already at 2 h p.i. of 68Ga-NOTA-PEG3-RM26. Results of this study suggest that variation in the length of the PEG spacer can be used for optimization of targeting properties of peptide-chelator conjugates. However, the influence of the mini-PEG length on biodistribution is minor when di-, tri-, tetra- and hexaethylene glycol are compared.