Hypoxic regions in solid tumors are critically important in tumor physiology and cancer treatment, and they appear to be strongly associated with malignant progression and therapeutic resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) was recently reported to be a master transcriptional activator of various genes related to malignant phenotypes; therefore, noninvasive imaging of HIF-1-active tumors is important for targeted cancer therapy and predicting prognosis. The α-subunit of HIF-1 (HIF-1α) is degraded in an oxygen-dependent manner under normoxic conditions, whereas it is stable under hypoxic conditions and controls HIF-1 transcriptional activity. Thus, a probe that is degraded in a similar manner as HIF-1α can be used to evaluate HIF-1 activity in vivo. A fusion protein named POS was recently developed as such a probe. It consists of 3 domains: protein transduction, oxygen-dependent degradation, and monomeric streptavidin domains. The streptavidin moiety is labeled by ([123I]iodobenzoyl)norbiotinamide (123I-IBB), a radioiodinated biotin derivative, to produce 123I-IPOS. 123I-IPOS is stable in hypoxic cells and accumulates in tumors. Tumors could be clearly visualized 24 h after 123I-IBB injection. The tumoral accumulation of 123I-IPOS was positively correlated with HIF-1 transcriptional activity and coincided with the HIF-1-positive areas. Thus, 123I-IPOS is a potential probe for the imaging of HIF-1-active hypoxic regions in tumors. However, the drawback of 123I-IPOS was that a long time (24 h) was required to obtain a well-contrasted image. To overcome this problem, a pretargeting approach was used. In the pretargeting approach, a radionuclide is delivered separately from the tumor-seeking molecule, that is, POS is administered and allowed to undergo degradation in normal regions, after which 123I-IBB is administered. 123I-IBB is not retained in normal regions; however, it binds to the POS retained in HIF-1-active regions, which enables specific imaging of such regions. In fact, 123I-IBB accumulated in tumors pretargeted with POS, whereas it did not accumulate in regions that were not pretargeted. Tumors could be clearly visualized 6 h after injection, which is one-fourth of the time required for the direct targeting approach. The tumoral accumulation of 123I-IBB in POS-pretargeted mice was positively correlated with HIF-1 transcriptional activity and coincided with the HIF-1-positive areas. Thus, POS pretargeting with 123I-IBB is a useful technique for the rapid imaging and detection of HIF-1-active regions in tumors.