Carbonic anhydrase IX (CA IX) is the first carbonic anhydrase found to be associated with cancer that is over expressed in a variety of human solid tumors. As a surrogate marker for hypoxia, the expression of CA IX is strongly upregulated in hypoxic tumors by hypoxia and hypoxia-inducible factor 1a (HIF-1a). In our pursuit of a CA IX-specific PET probe, we designed and synthesized a peptide-based CA IX imaging probe by the efficient click reaction of 1,3-dipolar cycloaddition of terminal alkynes and organic azides. The probe F-18-CA IX-P1-4-10 was obtained with a radiochemical yield of 35-45% (n=5) and radiochemical purity of > 99% in 70-80 min (HPLC purification time included). F-18-CA IX-P1-4-10 had good stability in phosphate buffered saline (PBS), but about 51% peptide degradation was detected in new-born calf serum (NBCS) after incubation. Preliminary microPET/CT experiments demonstrated a specific uptake of F-18-CA IX-P1-4-10 in HT29 tumor and the uptake of F-18-CA IX-P1-4-10 was blocked by peptide CA IX-P1-4-10-Yne pretreatment. Immunohistochemical staining and western blotting studies confirmed the HT29 tumor was CA IX-positive which further proved tumor accumulation of F-18-CA IX-P1-4-10 was correlated with CA IX expression. The results suggest that F-18-CA IX-P1-4-10 is a promising PET tracer for the specific imaging of CA IX-expressing tumors at the molecular level.