[en] Highlights: • We developed a radioiodinated peptide probe targeting αvβ6 integrin (¹²³I-IFMDV2). • ¹²³I-IFMDV2 had a high affinity and selectivity for αvβ6 integrin. • ¹²³I-IFMDV2 showed a specific binding to αvβ6 integrin in vivo. • ¹²³I-IFMDV2 enabled clear visualization of the αvβ6-integrin-positive tumor. - Abstract: Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT). Methods: Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed. Results: Among the 4 probes examined in this study, ¹²⁵I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, ¹²³I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft. Conclusion: ¹²³I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC