The present study aimed to label Z(HER2:V2) with technetium-99m (Tc-99m) using a simple method and to evaluate its clinical potential as a diagnostic probe for human epidermal growth factor receptor type 2 (HER2)-positive tumors. The ZHER2:V2 (Affibody molecule of ZHER2:2395-C, which is based on the ZHER2:342 binding sequence with C-terminal engineered cysteine) with C-terminal chelating sequence GGGC was designed and labeled with 99mTc. The 99mTc-ZHER2:V2 labeling efficiency was analyzed. The cellular uptake, retention and binding affinity, and the stability of the probe were examined in vitro. 99mTc-ZHER2:V2 biodistribution analysis and imaging were performed in BALB/c nude mice bearing SKOV3 (HER2-overexpression) xenografts. Furthermore, imaging of the probe was performed in MCF-7 (HER2 low-expression) xenografts. The 99mTc-ZHER2:V2 labeling efficiency was identified as 98.99 +/- 0.99% (n=6), and was stable in physiological saline and fresh human serum at 37 degrees C in vitro. The cellular uptake peak of SKOV3 cells at 24 h was 6.15 +/- 0.18%, the cellular retention ratio of the probe was 48.58 +/- 4.52% at 6 h following interrupted incubation, and similar to 70% of 99mTc-ZHER2:V2 was membrane bound following 24 h. 99mTc-ZHER2:V2 was blocked by excess amounts of unlabeled ZHER2:V2 in SKOV3 cells. 99mTc-ZHER2:V2 exhibited high distribution (10.07% ID/g) in SKOV3 xenografts at 6 h following injection. The single photon emission computed tomography (SPECT) imaging revealed clear localization of 99mTc-ZHER2:V2 in the SKOV3 xenografts at 4 h. However, there was low uptake in MCF-7 tumors on the SPECT images. The SKOV3 xenograft imaging could be blocked by excess amounts unlabelled ZHER2:V2. 99mTc-ZHER2:V2 is an easy and quick labeling method, with high labeling yields, and radiochemical purity. 99mTc-ZHER2:V2 is a promising probe for the diagnosis of HER2-overexpression tumors and the monitoring of therapy response.