Titin is a 3000 kDa large protein of vertebrate striated muscle which extends from Z discs to M lines. Within the segment of titin that locates in the I band, tissue-specific isoforms are expressed by differential splicing in correlation to the sarcomeric ultrastructure. We have now searched the M-line region of titin for differential expression. The 20 kb section from the 3' end of the gene has been sequenced and contains 23 exons. Exon/intron organization is correlated to the modular organization of the titin protein. The six exons at the 3' end of the gene encode the M-line section of titin and are referred to as Mex1 to Mex6. Analysis of the RNAs expressed in different rabbit striated muscles reveals that the exon Mex5 is either included or excluded in the titin mRNA during splicing. The levels of inclusion of Mex5 vary between different types of striated muscles. Heart expresses (Mex5+)-titin, skeletal muscles co-express tissue-specifically distinct ratios of (Mex5+) and (Mex5-)-titins. In situ hybridization of whole-mount mouse embryos with Mex5 antisense RNA provide no evidence for the exclusion of Mex5 during embryonic development. We speculate that the establishment of differential splicing pathways of M-line titin late during development may correlate with and explain the postnatal development of different M-line fine structures in the different muscles. Comparison of titin gene sequences from different vertebrates reveals that the intron sequences located upstream of Mex3 and Mex5, referred to as Min-2 and Min-4, respectively, have remained strongly conserved during evolution. While the conservation of Min-4 may be explained by its participation in the regulation of the differential skipping of Mex5, the functional significance of the conservation of the Min-2 intron located upstream of Mex3 is yet unknown.