The development of a suitable radioligand for in vivo targeting CCK-2 receptor expressing tumours, such as medullary thyroid carcinoma, is a major recent focus in the field of radiopharmacy. Initial developments using [DGlu1]minigastrin (MG0) derivatives showed high uptake by the kidneys resulting as the dose limiting organ for radiopeptide therapy. In this study we describe the comparison of the optimised shortened peptide analogues, [DOTA0,DGlu1,desGlu2-6]minigastrin (DOTA-minigastrin-11) with [DOTA0,DGlu1,desGlu2-6,Ile11]minigastrin (DOTAilegastrin), labelled with 111In and 68Ga. In vitro tests included stability in plasma and plasma protein binding. Internalisation and receptor binding assays using a CCK-2 receptor positive cell-line (AR4-2J, rat pancreatic tumour cells) were performed and biodistribution in AR4-2J tumour bearing nude mice was studied. DOTA-ilegastrin showed a superior stability in plasma in comparison with DOTA-minigastrin-11. 68Ga/111In-labelled DOTA-ilegastrin revealed, however, significantly reduced internalisation and lower receptor affinity than DOTAminigastrin- 11. For both radiologiands rapid renal excretion and low unspecific retention in most organs was observed. Tumour uptake of 111In/68Ga-DOTA-minigastrin-11 was much higher (4.9 and 5.7 %ID/g 1 h p.i.) compared to 111In/68Ga- DOTA-ilegastrin (1.2 and 1.0 %ID/g). Kidney retention was clearly lower for the short chain analogues as compared to MG0 derivatives. Tumour uptake could be reduced by more than 60% by coinjection of 50 μg human minigastrin. DOTA-minigastrin-11 showed considerably lower kidney uptake, but also higher metabolic instability as compared to MG0 derivatives. No relevant differences between 111In- and 68Ga-labelled derivatives were observed. Replacement of methionine with isoleucine lead to dramatic loss of receptor affinity.