Titin is the largest protein known, and is essential for organising muscle sarcomeres. It has many domains with a variety of functions, and stretches from the Z-line to the M-line in the muscle sarcomere. Close to the M-line, titin contains a kinase domain, which is known to phosphorylate the Z-line protein telethonin in developing muscle (Mayans, O., van der Ven, P. F., Wilm, M., Mues, A., Young, P., Furst, D. O., Wilmanns, M. and Gautel, M. (1998) Nature 395, 863-869). This phosphorylation is thought to be important for initiating or regulating myofibrillogenesis. We used a gene-targeting approach in cultured myoblasts to truncate the titin gene so that the kinase domain and other domains downstream of the kinase were not expressed. We recovered cells in which one allele was targeted. We found that these cells expressed both the full-length and a truncated titin that was approximately 0.2 MDa smaller than the corresponding band from wild-type cells. Myofibrillogenesis in these cells was impaired, in that the myotubes were shorter, and the organisation of the muscle sarcomeres, M- and Z-lines was poorer than in wild-type cells. There was also an overall reduction in levels of titin and skeletal myosin expression. These results suggest that the activity of the titin kinase domain and downstream sequence are important in organising myofibrils both at the M- and the Z-line early in myofibrillogenesis.