G protein-coupled receptor agonists are being used as radiolabeled vectors for in vivo localization and therapy of tumors. Recently, somatostatin-based antagonists were shown to be superior to agonists. Here, we compare the new 111In/68Ga-labeled bombesin-based antagonist RM1 with the agonist 111In-AMBA for targeting the gastrin-releasing peptide receptor (GRPR). IC50, Kd values, and antagonist potency were determined using PC-3 and HEK-GRPR cells. Biodistribution and imaging studies were done in nude mice transplanted with the PC-3 tumor. The antagonist potency was assessed by evaluating the effects on calcium release and on receptor internalization monitored by immunofluorescence microscopy. The IC50 value of (nat)In-RM1 was 14 +/- 3.4 nmol/L. (nat/111)In-RM1 was found to bind to the GRPR with a Kd of 8.5 +/- 2.7 nmol/L compared with a Kd of 0.6 +/- 0.3 nmol/L of 111In-AMBA. A higher maximum number of binding site value was observed for 111In-RM1 (2.4 +/- 0.2 nmol/L) compared with 111In-AMBA (0.7 +/- 0.1 nmol/L). (nat)Lu-AMBA is a potent agonist in the immunofluorescence-based internalization assay, whereas (nat)In-RM1 is inactive alone but efficiently antagonizes the bombesin effect. These data are confirmed by the calcium release assay. The pharmacokinetics showed a superiority of the radioantagonist with regard to the high tumor uptake (13.4 +/- 0.8% IA/g versus 3.69 +/- 0.75% IA/g at 4 hours after injection. as well as to all tumor-to-normal tissue ratios. Despite their relatively low GRPR affinity, the antagonists 111In/68Ga-RM1 showed superior targeting properties compared with 111In-AMBA. As found for somatostatin receptor-targeting radiopeptides, GRP-based radioantagonists seem to be superior to radioagonists for in vivo imaging and potentially also for targeted radiotherapy of GRPR-positive tumors.