Titin (also called connectin) is a megadalton protein that constitutes up to 10% of the myofibrillar protein in skeletal and cardiac muscles. The objectives of this study were: (1) to determine a portion of the primary structure of titin; and (2) to identify interactions of cloned titin fragments with other myofibrillar proteins;A 0.2 kb cDNA clone, initially isolated by antibody screening of a chick cardiac muscle cDNA library, was used to screen a cDNA library constructed from 16-day embryonic chick cardiac muscle RNA size-selected for large RNAs. Three overlapping clones, containing 2.4 kb of titin sequence, were isolated. Immunoblots and immunofluorescence on myofibrils, with polyclonal titin antibodies and antibodies affinity purified against fusion protein encoded by one clone, demonstrated that this cDNA sequence codes for the region of titin located in the A-band, adjacent to the A-I junction, in myofibrils. Northern blots demonstrated hybridization to a very large RNA (>23 kb) expressed only in striated muscles. Southern blots suggested that titin is encoded by a single copy gene in chickens. The derived amino acid sequence showed the presence of two, 100-amino acid motifs, I and II, which are members of the fibronectin type III and immunoglobulin C2 families, respectively. These motifs are found in the pattern I-I-II-I-I-I-II-I-I;Cloned titin fragments of different length and motif composition were expressed in E. coli, and interactions of these fragments with selected myofibrillar proteins were examined. Results of solid-phase binding assays demonstrated that titin fragments bound to myosin, and, with lower affinity, to actin, but did not bind to tropomyosin or troponin-C. Binding to myosin or actin increased with decreasing salt concentration. Stronger interactions with myosin were observed with fragments containing more motifs or an intact copy of motif II. Competition assays demonstrated reduced affinity for myosin filaments that for monomeric myosin;In summary, the results of this study have provided a partial primary sequence of chicken muscle titin, from a previously uncharacterized portion of the molecule. This portion of titin is capable of binding to both myosin and actin.