Tenascin-X (TNX) is an extracellular matrix protein that is prominent in the heart and muscle. We previously showed that TNX is expressed in fibroblast cells in culture. To elucidate the molecular basis of the TNX gene expression, the promoter region of the mouse TNX gene (mTnx) has been characterized. The two adjacent transcription initiation sites were identified at 68 and 67 bp upstream of the previously known 5'-untranslated exon. Transient transfection of L and 293T cells with 5'-deletion constructs of the promoter region linked to the luciferase reporter revealed that the region (-141 to -136) containing a transcription factor Spl-binding element contributes to the expression of mTnx Site-directed mutagenesis of the Sp1-binding region confirmed this result. Electrophoretic mobility shift analysis using nuclear extracts obtained from the cells demonstrated that a distinct Sp1-DNA complex is formed at the element. Our results show that Spl plays a critical role in the gene expression of mTnx.