Fast skeletal muscle myosin light-chain I (MLC1f) and myosin light-chain 3 (MLC3f) mRNAs are both derived from a single rat MLC1/3f gene. MLC1f mRNA begins at the first exon of the gene, while MLC3f mRNA begins with exon 2, 10 kilobases downstream. Both mRNAs require alternate splicing of internal exons for accurate expression. We showed that a truncated rat MLC1f/3f gene lacking exon 1 and the first 6.3 kilobases of the intron separating exons 1 and 2 produced rat MLC3f mRNA in a developmentally regulated manner after introduction into myogenic mouse cells, thus demonstrating in vivo the presence of a functional promoter associated with exon 2. Correctly spliced mRNA was produced after transfer of this truncated gene into both myogenic and nonmyogenic cells, indicating that the pattern of splicing of this complex transcript was due to a structural features of the RNA and was independent of cell type.