<i>Objectives:</i> Tenascin-C (TNC) is an oligomeric glycoprotein of the extracellular matrix that is prominently expressed in malignant tumors. The purpose of this study was: (1) to determine the in vitro TNC splicing pattern in cultured human chondrocytes and chondrosarcoma cells, (2) to determine the in vivo TNC splicing pattern in clinical chondrosarcoma specimens, and (3) to perform survival analysis based on the TNC splicing pattern of the tumor specimens. <i>Methods:</i> Human articular chondrocytes and chondrosarcoma cells (cell line <i>JJ012</i>) were grown in a three-dimensional alginate bead system and harvested at two time points. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the in vitro TNC splicing pattern for the two cell types. Clinical chondrosarcoma specimens were obtained intra-operatively and underwent RT-PCR to determine the in vivo TNC splicing pattern. Specific immunohistochemical staining for the large TNC splice variant was performed on the clinical specimens. Survival analysis was used to determine the association between the specific TNC splicing pattern and survival. <i>Results:</i> The in vitro mRNA expression pattern of TNC in normal human articular chondrocytes was characterized by a high ratio of the small to the large splice variant (TNC<sub>small</sub>:TNC<sub>large</sub>), whereas the in vitro mRNA expression pattern for cultured chondrosarcoma cells was characterized by a low TNC<sub>small</sub>:TNC<sub>large</sub> ratio. Clinical chondrosarcoma specimens with a lower TNC<sub>small</sub>:TNC<sub>large</sub> ratio showed a trend towards decreased survival. The TNC splicing pattern of these specimens was verified through specific immunohistochemical staining for the large TNC isoform. <i>Conclusions:</i> The specific TNC splicing pattern may have clinical significance in chondrosarcoma. TNC expression may therefore play a future role in objecitve tumor grading and novel therapeutic approaches to this malignancy.