Using a Drosophila cell-free system, we have analyzed the regulation of alternative splicing of Drosophila muscle myosin heavy chain (MHC) transcripts. Splicing of MHC 3' end transcripts results in exclusion of adult-specific alternative exon 18, as is observed in embryonic and larval muscle in vivo. Mutations that strengthen either the 5' or the 3' splice sites of exon 18 do not promote inclusion of this exon. However, strengthening both splice junctions results in efficient removal of both introns and completely inhibits skip splicing. Our data suggest that the affinity of exons 17 and 19, as well as failure of constitutive splicing factors to recognize exon 18 splice sites, causes the exclusion of exon 18 in wild-type transcripts processed in vitro.