This paper analyses the HIV-1 gp120 epitope specificity and activation mechanisms (i.e., polyclonal versus oligoclonal) of antibodies present in the sera of alloimmune mice and humans. Sera from CBA mice engrafted with C57BL/6 lymphoid cells significantly reacted against the gp120-derived peptide as 261-270, which shares high homology with the membrane-proximal domain of HLA class II beta-chains (HLA/ gp120) and against the HIV gp120 V3 loop-derived peptides DP32 (HIV-1 MN-derived as 302-334) and C53 (HIV-1 IIIB-derived as 304-318). The same sera also reacted against the HIV-unrelated peptide necdin. Moreover, sera from BALB/c mice injected with LPS presented antibodies reacting against both HIV-related and -unrelated peptides, suggesting that similar mechanisms are shared in alloimmune and LPS-treated mice. A similar analysis was then performed on the sera of patients affected with beta-thalassemia major, receiving at least 10 blood transfusions/year. In particular, 15 of 58 (26%) sera from HIV-uninfected thalassemic patients showed a significantly reactivity against the HLA/gp 120-derived peptides. Moreover, 22 of 58 (38%) sera from the same cohort showed a significant reactivity against DP32 peptide. This reactivity was related to a polyclonal activation mechanism since the DP32-reactive sera significantly bound a panel of HIV-unrelated peptides, as observed by testing 22 sera against necdin, 21 against HSP65 kDa, 21 against amyloid-1, and 17 against MAGE-1 peptides. Moreover, a significant increase of IgG concentration was also observed in all thalassemic sera, when compared to healthy controls, without regard to the anti-gp120 antibody reactivity. Taken together, these results indicate that (i) allogeneic stimuli may induce anti-gp120 antibodies in CBA and in 38% of polytransfused patients and (ii) this reactivity is related to a polyclonal activation mechanism but not to a heightened concentration of IgG.