Carbonic anhydrase (CA) IX is a transmembrane protein overexpressed in many frequently occurring tumors associated with tumor hypoxia. Sulfonamides and their bioisosteres are known to inhibit CA IX activity. In this study, 4-(2-aminoethyl)benzenesulfonamide was conjugated to a tridentate ligand, N-2-picolyl- N-acetic acid and labeled with a 99mTc( I)-tricarbonyl moiety resulting in 99mTc(CO) 3 (L) (L= N-(pyridin-2-yl-methyl)- N2-(4-sulfamoylphenyl)-ethylaminoethyl acetate) complex, 99mTc- 5. Similarly the corresponding rhenium congener (Re- 4) was synthesized. The in vitro CA IX affinity and inhibitory activity of Re- 4 were determined and 99mTc- 5 was evaluated as a tracer for in vivo visualisation of CA IX expression. Evaluation of the in vitro affinity (inhibition constant, K i) of Re- 4 for CA isozymes I, II, IX and XII was carried out by assaying the CA catalyzed CO 2 hydration activity and efficacy studies were performed in HT 29 cell lines expressing CA IX under normoxia or hypoxia. Biodistribution studies of 99mTc- 5 were performed in xenograft mice bearing CA IX expressing tumors. The in vitro affinity of Re- 4 for CA IX was 58 nM and CA IX induced acidification of extracellular medium was efficiently reduced ( P<.05) in the presence of 1 mM Re- 4. Biodistribution studies indicated a maximal tumor uptake of 99mTc- 5 of 0.1% ID/g at 30 min post injection. 99mTc- 5 and its rhenium congener were synthesized and characterized. In vitro studies showed that the rhenium compound has a high affinity for CA IX and effectively inhibits CA IX activity. In vivo studies revealed a limited tracer accumulation in a CA IX expressing tumor but with increasing tumor-to-blood activity ratios as a function of time.