The 3' penultimate exon (exon 18) of transcripts from the muscle myosin heavy chain (MHC) gene of Drosophila melanogaster is excluded from mRNAs of embryonic and larval muscle, while it is included in mRNAs of adult thoracic muscles. By transforming organisms with the MHC gene 5' end, linked in-frame to the MHC gene 3' end, we were able to generate correct tissue-specific expression of this minigene and stage-specific splicing of exon 18, indicating that all the cis-acting sequences necessary for alternative splicing are contained within the construct. The 3' splice site that precedes exon 18 is unusually purine-rich, may form a stem-loop structure with the 5' splice site following exon 18, and is conserved relative to the splice site of an alternative exon of the Drosophila alkali myosin light chain gene. We converted the MHC gene 3' splice junction to a consensus splice site and also inserted the branchpoint and 3' splice site of a constitutively spliced intron in its place. These alterations had no effect on the splicing pathway in vivo, ruling out the possibility that the unusual splice junction, or secondary structures that involve this splice junction, directly regulate alternative splicing of exon 18.