Large conductance voltage and calcium-activated potassium (BKCa) channels link electrical excitability with cellular signalling. A single gene (KCNMA1 or slo) encodes the α-subunits that form the native BKCa channel pore. Alternative pre-mRNA splicing of the α-subunits determines the channel phenotype, including its intrinsic voltage dependence and calcium sensitivity (Chen et al., 2005). The identification and characterization of BKCa channel splice variants depends on the use of low throughput patch clamp electrophysiology. Here we present a robust, high throughput assay to screen BKCa splice variants, which was initially optimized using the voltage-sensitive dye Membrane Potential Red (Molecular Probes),the automated fluid-handling fluorometer FlexStation II ®, and HEK 293 cells transiently or stably transfected with the stress-related exon (STREX) BKCa channel splice variant. As BKCa channels are activated by elevation of intracellular free calcium, ionomycin (a calcium ionophore) was used to stimulate channel activity via influx of calcium ions. Ionomycin (1µM) induced a significant prolonged hyperpolarisation in HEK cells transfected with the STREX variant but not in untransfected controls. The hyperpolarizing response was attenuated by the removal of extracellular calcium and was blocked by the BKCa channel inhibitor paxilline with an IC50 of 0.23 ± 0.05 µM (n=15). The robustness of the assay allowed us to ask whether we could discriminate between BKCa channel splice variants with known differences in calcium sensitivities. To test this we used the STREX, ZERO, and e22 splice variants that differ in the alternatively spliced inserts present at splice site 2 in the c-terminus of the α-subunit and as a result display a range of calcium sensitivities. In accordance with previous electrophysiological assays the voltage-sensitive dye assay revealed that expression of the STREX variant produced the greatest hyperpolarizing response to ionomycin. e22 and ZERO produced significantly smaller responses, of 64 ± 17% and 45 ± 13% respectively, when compared to the STREX response (taken as 100%) with the rank order in agreement with electrophysiology assays (n=8, data are mean ± sem p<0.001 ANOVA with Tukey post-hoc test). Expression of the different BKCa channel splice variants had no significant effect on the ionomycin-induced calcium influx in HEK293 cells as measured using Calcium FLIPR 3A (Molecular Probes). This approach allowed reproducible high throughput data to discriminate between BKCa channel splice variants that can be applied to the analysis of BKCa channel splice variant regulation, cell surface expression and pharmacology.