The envelope glycoproteins G1/G2 of Toscana virus (TOSV) seem to have the most important protective role in stimulating antibodies against the disease in humans, as well as antibodies against the Nucleoprotein (N), a partial neutralizing activity. Mice immunized with TOSV recombinant Nucleoprotein developed a strong humoral response to the TOSV that revealed the presence of neutralizing antibody than in vitro assay. The neutralizing antibody titre of mice immunized with the whole TOSV was analyzed before and after absorption of the sera with the recombinant N protein. A decrease of the neutralizing activity was observed in the treated sera. Similar results were obtained absorbing human anti-TOSV positive sera with the recombinant N protein. This study was designed to identify the nature of antibodies produced against the N protein of TOSV in mice and to establish correlation with antibodies produced in humans by natural infection.