Abstract Purpose Radiolabeled somatostatin analogues are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin-receptor-positive tumors. The aim of this study was to evaluate a new somatostatin analogue designed for the labeling with 99mTc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC 0), 1-Nal 3, Thr 8]-octreotide ([HYNIC]-NATE), using ethylenediamine- N, N′-diacetic acid (EDDA) and tricine as coligands. Methods Synthesis was preformed on a solid phase using a standard Fmoc strategy. Labeling with 99mTc was performed at 100°C for 10 min using SnCl 2 as a reductant. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate affinity was determined in AR4-2J cell membranes. The internalization and externalization rates were studied in sstr 2-expressing AR4-2J cells. Biodistribution of radiopeptide was studied in rats bearing the AR4-2J tumor. Results Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. Peptide conjugate showed high affinity binding for sstr 2. The radioligand showed a moderate and specific internalization into AR4-2J cells (14.13±0.61% at 4 h). In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in somatostatin-receptor-positive organs. After 4 h, uptake in the AR4-2J tumor was 1.33±0.23%ID/g (percentage of injected dose per gram of tissue). Conclusion These data show that [ 99mTc/EDDA/tricine/HYNIC]-NATE is a specific radioligand for the somatostatin-receptor-positive tumors and is a suitable candidate for clinical studies.