Developmental changes in the alternative splicing patterns of titin were observed in rat cardiac muscle. Titin from 16-day fetal hearts consisted of a single 3710kDa band on SDS agarose gels, and it disappeared by 10 days after birth. The major adult N2B isoform (2990kDa) first appeared in 18-day fetal hearts and its proportion in the ventricle increased to approximately 85% from 20 days of age and older. Changes in three other intermediate-sized N2BA isoform bands also occurred during this same time period. The cDNA sequences of fetal cardiac, adult ventricle, and adult soleus were different in the PEVK and alternatively spliced middle Ig domain. Extensive heterogeneity in splice patterns was found in the N2BA PEVK region. The extra length of the fetal titin isoforms appeared to be due to both a greater number of middle Ig domains expressed plus the inclusion of more PEVK exons. Passive tension measurements on myocyte-sized fragments indicated a significantly lower tension in neonate versus adult ventricles at sarcomere lengths greater than 2.1μm, consistent with the protein and cDNA sequence results. The time course of the titin isoform switching was similar to that occurring with myosin and troponin I during development.