To image hypoxia-inducible factor-1 (HIF-1)-active tumors, we previously developed a chimeric protein probe ([123/125I]IPOS) that is degraded in the same manner as HIF-1α under normoxic conditions. In the present study, we aim to show that the accumulation of radioiodinated POS reflects the expression of HIF-1. In vivo single-photon emission computed tomography (SPECT)/X-ray CT (CT) imaging, autoradiography, and double-fluorescent immunostaining for HIF-1α and pimonidazole (PIMO) were carried out 24 h after the injection of [125I]IPOS. Tumor metabolite analysis was also carried out. A tumor was clearly visualized by multi-pinhole, high-resolution SPECT/CT imaging with [125I]IPOS. The obtained images were in accordance with the corresponding autoradiograms and with the results of ex vivo biodistribution. A metabolite analysis revealed that 77% of the radioactivity was eluted in the macromolecular fraction, suggesting that the radioactivity mainly existed as [125I]IPOS in the tumors. Immunohistochemistry revealed that the HIF-1α-positive areas and PIMO-positive areas were not always identical, only some of the regions were positive for both markers. The areas showing [125I]IPOS accumulation were positively and significantly correlated with the HIF-1α-positive areas (R = 0.75, P < 0.0001). The correlation coefficient between [125I]IPOS-accumulated areas and HIF-1α-positive areas was significantly greater than that between the [125I]IPOS-accumulated areas and the PIMO-positive areas (P < 0.01). These findings indicate that [125I]IPOS accumulation reflects HIF-1 expression. Thus, [123/125I]IPOS can serve as a useful probe for the molecular imaging of HIF-1-active tumors. (Cancer Sci 2011; 102: 2090–2096)