Integrin alpha(v)beta(6) is overexpressed in a variety of cancers, and its expression is often associated with poor prognosis. Therefore, there is a need to develop affinity reagents for noninvasive imaging of integrin alpha(v)beta(6) expression since it may provide early cancer diagnosis, more accurate prognosis, and better treatment planning. We recently engineered and validated highly stable cystine knot peptides that selectively bind integrin alpha(v)beta(6) with no cross-reactivity to integrins alpha(v)beta(5), alpha(5)beta(1), or alpha(v)beta(3), also known to be overexpressed in many cancers. Here, we developed a single photon emission computed tomography (SPECT) probe for imaging integrin alpha(v)beta(6) positive tumors. Cystine knot peptide, S(0)2, was first conjugated with a single amino acid chelate (SAAC) and labeled with [Tc-99m(H2O)(3)(CO)(3)](+). The resulting probe, Tc-99m-SAAC-S(0)2, was then evaluated by in vitro cell uptake studies using two alpha(v)beta(6) positive cell lines (human lung adenocarcinoma cell line HCC4006 and pancreatic cancer cell line BxPC-3) and two alpha(v)beta(6) negative cell lines (human lung adenocarcinoma cell line H838 and human embryonic kidney cell line 293T). Next, SPECT/CT and biodistribution studies were performed in nude mice bearing HCC4006 and H838 tumor xenografts to evaluate the in vivo performance of Tc-99m-SAAC-S(0)2. Significant differences in the uptake of Tc-99m-SAAC-S(0)2 were observed in alpha(v)beta(6) positive vs negative cells (P < 0.05). Biodistribution and small animal SPECT/CT studies revealed that Tc-99m-SAAC-S(0)2 accumulated to moderate levels in antigen positive tumors (similar to 2% ID/g at 1 and 6 h postinjection, n = 3 or 4/group). Moreover, the probe demonstrated tumor-to-background tissue ratios of 6.81 +/- 2.32 (tumor-to-muscle) and 1.63 +/- 0.18 (tumor-to-blood) at 6 h postinjection in alpha(v)beta(6) positive tumor xenografts. Co-incubation of the probe with excess amount of unlabeled S(0)2 as a blocking agent demonstrated significantly reduced tumor uptake, which is consistent with specific binding to the target. Renal filtration was the main route of clearance. In conclusion, knottin peptides are excellent scaffolds for which to develop highly stable imaging probes for a variety of oncological targets. Tc-99m-SAAC-S(0)2 demonstrates promise for use as a SPECT agent to image integrin alpha(v)beta(6) expression in living systems.