The differential diagnosis of chordoma includes chondrosarcoma and ependymoma. We describe four cases of chordoma characterized by enzyme histochemistry in plastic-embedded sections. All the chordomas exhibited strong 5'-nucleotidase positivity localized on the plasma membrane. None of the fetal notochord remnants, chondrosarcomas, ependymomas, or chondroid chordomas tested showed such a reaction. The lack of similarity in enzyme staining between fetal notochord and chordomas is unexpected since notochord has been traditionally regarded as the source of chordomas. This staining pattern provides a marker that can be useful in differentiating chordoma from other neoplasms which have a similar appearance by light microscopy.