There is currently no simple method to detect the antigen specificity of anti-HIV-1 IgG intrathecal synthesis (IS). Fifty-seven pairs of serum and corresponding CSF from 29 HIV-1 seropositive patients were adjusted to an identical concentration of total IgG and tested by a commercial HIV-1 Western Blot (WB) assay. IgG IS to a given HIV-1 protein was demonstrated when the corresponding band was present in CSF but absent or significantly less represented in serum. A total anti-HIV-1 IS was defined as the presence of an IS to one or more HIV-1 antigens. Our WB analysis of CSF and serum, compared with conventional mathematical formulas, showed a higher sensitivity in demonstrating anti-HIV-1 IgG IS. Moreover, the method disclosed which HIV-1 proteins represent the target of IgG IS. This procedure is easy to perform and therefore may represent a valuable tool to study central nervous system (CNS) involvement by HIV-1 during different stages of infection.