In the present work we investigate whether artificial alterations of the structure of an inactive retrovirus-encoded protein could transform it in a superantigen. As a model system we used a recombinant human immunodeficiency virus (HIV)-1 p24 protein and two of its variants in which a short peptide corresponding to sequences of gp41 of HIV-1 (HIV-1 p24*) or gp36 of HIV-2 (HIV-1-2 p24*) has been inserted nearby the carboxy-terminal end of HIV-1 p24. As expected both HIV-1 p24 and HIV-1 p24* were inactive, while HIV-1-2 p24* was a potent inducer of human, but not murine, T cell proliferation. The possibility that the observed activity was due to contaminants was ruled out since the proliferative response could be specifically inhibited by a monoclonal anti-p24 antibody and by a peptide encompassing the area of HIV-1 p24/HIV-2 gp36 junction. Furthermore, the data exclude the possibility that the gp36 insertion is per se responsible for the observed proliferative activity. The analysis of the functional, phenotypic and molecular properties of the responding cells demonstrated that the response was class II dependent and that the activated cells were predominantly CD4+CD8- expressing a strongly biased repertoire of TCRBV segments. Collectively, these data strongly suggest that the HIV-1-2 p24* fusion protein shares common functional properties typical of superantigen molecules. Thus, our demonstration that a viral protein can be transformed into a superantigen simply by the insertion of a short peptide at the carboxy-terminal end has important implications for understanding the mode of action of retrovirus-encoded superantigens.