We have isolated a cDNA encoding mouse tenascin-X (TN-X), a new member of the family of tenascin genes. The TN-X gene lies in the major histocompatibility complex (MHC) class III region, as it is the case for its human counterpart. On Northern blots we detected a TN-X mRNA of approximately 13 kb in most tissues analyzed, whereas in various mouse cell lines mRNAs of approximately 11 and 13 kb were detected, suggesting the possibility of alternative splicing of TN-X transcripts. We raised antibodies against mouse TN-X fragments expressed in bacteria and used these antibodies to identify the TN-X protein in heart cell extracts and in the conditioned medium of a renal carcinoma cell line. The subunit molecular size of TN-X is approximately 500 kD, suggesting that the protein may contain up to 40 fibronectin type III repeats, making it the largest tenascin family member known yet. TN-X in conditioned medium, as well as the purified protein bind to heparin, but no binding to tenascin-C (TN-C), fibronectin, laminin or collagens could be detected. Thus the heparin-binding activity may be a common feature of the tenascins. The TN-X mRNA as well as the protein are predominantly expressed in heart and skeletal muscle, but the mRNA is found in most tissues at a low level. Immunostaining showed the protein to be associated with the extracellular matrix of the muscle tissues and with blood vessels in all of the tissues analyzed. Although the TN-X gene lies in the MHC class III locus, it is not expressed in the lymphoid organs analyzed, except for the staining around blood vessels. In skin and tissues of the digestive tract often a reciprocal distribution of TN-X and TN-C was observed.