Using the yeast two-hybrid system, we have recently reported that skeletal muscle-specific calpain, p94, binds specifically to connectin (or titin), a gigantic muscle elastic protein. Connectin has at least two binding sites for p94; one is at the N2-line region and the other is at the extreme C-terminus. In order to analyze the interaction between p94 and the C-terminus of connectin, we examined the C-terminal sequence of human skeletal muscle connectin. The sequence was essentially identical to that of heart muscle reported by Labeit and Kolmerer (1995, Science 270, 293-296), and the minimal binding site for p94 contained two IgC2 motifs and the intervening sequence called "M-is7." The exon encoding M-is7 is reported to be alternatively spliced depending on muscle tissues, resulting in the existence of both types of connectin with and without M-is7. However, the C-terminal region of connectin bound to p94 through M-is7. Our results suggest that the interaction between p94 and the C-terminus of skeletal muscle-type connectin is involved in tissue-specific myofibriogenesis.