In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three 99mTc-labeled gastrins of varying peptide chain length: 99mTcSG6 (99mTc-N4-Gln1gastrin(1–17)), 99mTcDG2 (99mTc-N4-Gly4,DGlu5gastrin(4–17)) and 99mTcDG4 (99mTc-N4-DGlu10gastrin(10–17)). Mouse blood samples were collected 5min after injection of each of 99mTcSG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/−) xenografts at 4h postinjection (pi). 99mTcSG6 or 99mTcDG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for 99mTcDG2 control and PA animal groups were only included. Treatment of mice with PA induced significant stabilization of 99mTc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 99mTcDG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only 99mTcDG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of 99mTcDG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios. In situ NEP/ACE-inhibition diversely affected the in vivo profile of 99mTc-radioligands based on different-length gastrins. Truncated 99mTcDG4 exhibited overall the most attractive profile during combined NEP/ACE-inhibition in mouse models, providing new opportunities for CCK2R-expressing tumor imaging in man with SPECT.