Tenascin-X (TN-X) is the newest member of the tenascin family of extracellular matrix proteins and it is highly expressed in muscular tissues during development. To gain insight into the possible functions of TN-X during development, we evaluated its expression in the rat embryo. Using an 800 bp cDNA encoding the fibrinogen-like domain of TN-X, we show that TN-X expression begins in migrating cells of the epicardium in the E12 heart. The epicardium provides progenitors of fibrous and vascular tissue to the developing heart. After the epicardium is complete, TN-X is expressed in the sub-epicardial space in association with developing blood vessels, and later by non-myocytes dispersed through the myocardial wall. A similar pattern of TN-X expression, first in connective tissue surrounding muscle, and then by a subset of cells within muscle, was seen in para-axial, body wall, craniofacial, and appendicular muscle. This pattern suggests a role in connective tissue cell migration and late muscle morphogenesis. TN-X is also highly expressed in the interdigital space at E15 and surrounding developing tendons, suggesting an additional role in cell fate determination. Although the pattern of TN-X expression is distinct from that of tenascin C, they are frequently expressed in close proximity. Indirect genetic evidence in humans suggests an essential function for TN-X, and the pattern of TN-X expression in heart, skeletal muscle, and limb is consistent with this hypothesis. ©1995 Wiley-Liss, Inc.