Ecto-5'-nucleotidase (eN, CD73) mediates extracellular adenosine production from 5'-AMP. Non-enzymatic functions of eN have also been reported. The aim of the study was to investigate the role of ecto-5'-nucleotidase in aggressive melanoma behaviour. Analysis of the involvement of eN in adhesion, migration and invasion revealed eN functions unknown to date. We found that following eN blockade by concanavalin A, the strength of adhesion to different ECM proteins was not altered, but at the same time the invasion ability of the cells was decreased. On the other hand, knocking down eN in melanoma cells did not influence cell invasion but abolished their migration on tenascin C (TnC). Ecto-5'-nucleotidase seems to fulfil a more distinct role as a receptor than as an enzyme in the cell interaction and mobility on TnC. Ecto-5'-nucleotidase activates also focal adhesion kinase and enhances the formation of complexes upon cell adhesion to TnC. All these observations prove that an eN-TnC complex is involved in cell migration and invasion and thus in the regulation of melanoma progression.