The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo(4) mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types.