The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6al, 6a2, 6bl, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6bl, resulting in the failure to produce all exon 6bl-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.We have reported that the haplolethal (HL) region of the Shaker gene complex (ShC) of Drosophila melanogaster (13) harbors the structural gene for troponin I (TnI) (2) and ascribed the heldup (hdp) mutations, originally described by Deak (10), to the TnI gene on the basis of genetic complementation tests. Later, Beall and Fyrberg (4) showed that some hdp mutants lack several TnI isoforms. However, since the mutant phenotype consists in the severe depletion of selected muscles, it is not clear whether the lack of specific TnI isoforms is the cause or the consequence of the muscle defect.We find that the allele mutation hdp3 consists of a single nucleotide change at the A(G/G) 3' border of the intron preceding exon 6bl of TnI, which is converted to A(A/G). The splicing complex in the mutant does not seem to recognize the new splice site and consequently does not express exon 6bl-containing RNA isoforms in any muscle, including those that do not show the mutant phenotype. The muscles not affected by the absence of exon 6bl are those in which the expression of this exon would have been quantitatively minor.TpI is a constituent of the thin filaments and plays a regulatory role in muscle contraction (33). It binds to actin, blocking the actin-myosin interaction in the resting state.Upon Ca2' entry, TnI shifts to interact with troponin C, leaving actin free and allowing the relative displacement of thin and thick filaments. Muscle physiology in insects shows peculiar features which sustain their extraordinary performance during wing beat (25). In the insect fibrillar flight muscles (DLM and DVM), calcium alone is not enough to produce full activation. The Ca2+-activated muscles are further activated in both tension and ATPase activity by small strains (1 to 3%) of their fibers. This property, first shown in the giant waterbug Lethocerus sp. (32), is also seen in D. melanogaster (23) and enables the wings to beat at high frequency. It is expected that the diversity of mechanical * Corresponding author. t Present address: