Strong overexpression of glucagonlike peptide-1 (GLP-1) receptors in human insulinoma provides an attractive target for imaging. The first clinical trials demonstrated that GLP-1 receptor SPECT/CT using Lys(40)(Ahx 6-aminohexanoic acid-DOTA-(111)In)NH(2)-exendin-4 can localize hardly detectable insulinomas. However, Lys(40)(Ahx-DOTA-(111)In)NH(2)-exendin-4 imaging has drawbacks related to the use of (111)In in that it is costly and carries a relatively high radiation burden for the patient. The aim of this study was the preclinical evaluation of Lys(40)(Ahx-DOTA-(68)Ga)NH(2)-exendin-4 for PET/CT and Lys(40)(Ahx-hydrazinonicotinamide HYNIC-(99m)Tc)NH(2)-exendin-4 for SPECT/CT. Internalization, biodistribution, dosimetry, and imaging studies were performed in the Rip1Tag2 mouse model of pancreatic beta-cell carcinogenesis and compared with our gold standard Lys(40)(Ahx-DOTA-(111)In)NH(2)-exendin-4. Poly-glutamic acid and Gelofusine, a gelatin-based plasma expander, were used for renal uptake reduction studies. The tumor uptake of Lys(40)(Ahx-DOTA-(68)Ga)NH(2)-exendin-4 was 205 +/- 59 percentage injected activity per gram of tissue at 4 h. Other GLP-1 receptor-positive organs showed more than 4.8 times lower radioactivity uptake. Lys(40)(Ahx-HYNIC-(99m)Tc/ethylenediaminediacetic acid EDDA)NH(2)-exendin-4, compared with its (111)In- and (68)Ga-labeled sister compounds, showed significantly less tumor and organ uptake. The significantly lower tumor and organ uptake of Lys(40)(Ahx-HYNIC-(99m)Tc/EDDA)NH(2)-exendin-4 did not result in inferior tumor-to-organ ratios or reduced image quality. All radiopeptides tested showed a high tumor-to-background ratio, resulting in the visualization of small tumors (maximum diameter between 1.0 and 3.2 mm) by SPECT and PET. The only exception was the kidneys, which also showed high uptake. This uptake could be reduced by 49%-78% using poly-glutamic acid, Gelofusine, or a combination of the 2. The estimated effective radiation dose was 3.7 muSv/MBq for Lys(40)(Ahx-HYNIC-(99m)Tc/EDDA)NH(2)-exendin-4, which was 8 times less than that for Lys(40)(Ahx-DOTA-(68)Ga)NH(2)-exendin-4 and 43 times less than that for Lys(40)(Ahx-DOTA-(111)In)NH(2)-exendin-4. These promising pharmacokinetic and imaging data show that Lys(40)(Ahx-DOTA-(68)Ga)NH(2)-exendin-4 and Lys(40)(Ahx-HYNIC-(99m)Tc/EDDA)NH(2)-exendin-4 are suitable candidates for clinical GLP-1 receptor imaging studies.