AbstractCarbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE₃-ZCAIX:2, was labeled with ¹¹¹In and characterized in vitro. Tumor-targeting properties of [¹¹¹In]In-DOTA-HE₃-ZCAIX:2 were compared head-to-head with properties of the parental variant, [^99mTc]Tc(CO)₃-HE₃-ZCAIX:2, and the most promising antibody fragment-based tracer, [¹¹¹In]In-DTPA-G250(Fab’)₂, in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the ^99mTc-labeled parental variant, [¹¹¹In]In-DOTA-HE₃-ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [¹¹¹In]In-DOTA-HE₃-ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [¹¹¹In]In-G250(Fab’)₂. In conclusion, [¹¹¹In]In-DOTA-HE₃-ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.