Alternative splicing of the Shaker (Sh) locus of Drosophila generates several transcripts with divergent 5' and 3' domains that produce kinetically distinct K+ currents in Xenopus oocytes. Although suggestive that alternative splicing may be involved in generating K+ channel diversity, clear tissue-specific differences in the distribution of particular Sh gene products have not been demonstrated. Using lacZ as a reporter gene for accurate splicing of variable Sh3' domains, we observe differences in beta-galactosidase expression patterns in transgenic animals that indicate both temporal and spatial regulation of 3' splice choice. The differences in 3'splice choice can account for variation in recovery kinetics of Sh-encoded K+ currents recorded in adult flies. The results indicate that tissue-specific expression of functionally distinct Sh K+ channels is regulated, in part, at the level of pre-mRNA splicing and implicate sequences in or around the 3' splice sites in regulating the choice of 3' domain.