K, channels and K+-coupled membrane transporters are important targets for drug discovery. We previously developed a triazacryptand (TAG)-based K+ sensor, TAG-Red, and demonstrated its utility to image K+ waves in mouse brain in vivo (Padmawar et al. Nat. Methods. 2005, 2, 825-827). Here, we synthesized a green-fluorescing dextran conjugate of TAC-bodipy ("TAC-Lime(dax)") for use as an extracellular K+ sensor and demonstrated its utility in measuring K+ transport across cell membranes. TAC-Lime(dex) fluorescence increased by 50% with increasing [K+] from 0 to 2 mM and was insensitive to [Na+], [Cl-], or pH. K+ efflux from cells was quantified from increasing extracellular TAC-Limedex fluorescence following cell immersion in K+-free buffer. In HT-29 cells, K+ efflux was 2.0 +/- 0.1 mu mol/cm(2)/s, increasing 8-fold following K+ channel activation by ATP: the increase in K+ efflux was inhibited by a K+ channel blocker or by preventing cytoplasmic calcium elevation. Electroneutral K+/Cl- cotransport was demonstrated in SiHa cells, in which K+ efflux was increased 3-fold by hypotonic challenge, the increase in K+ efflux was fully inhibited by a K+/Cl- transport blocker. K+ efflux measurements were adapted to a commercial fluorescence platereader for automated screening. The fluorescence-based K+ transport assay largely replaces assays requiring radioactive rubidium and is suitable for high-throughput identification of K+ transport modulators.