Optimal conditions for in vitro anti-human immunodeficiency virus type 1 (HIV-1) antibody (Ab) synthesis and detection were re-appraised. Western blot (WB) and radioimmunoassay (RIA) could detect about 1 and 10 ng, respectively, of HIV-1-specific Ab (HIV-Ab), while the sensitivity of an enzyme-linked immunosorbent assay (ELISA) was much lower. Optimal HIV-Ab recovery was obtained by culturing 2.5 x 10(6) peripheral blood mononuclear cells (PBMC)/ml from seropositive subjects for 16 days in the absence of mitogens; at higher cell concentrations, background levels were unacceptably high. The background of non-de novo synthesized HIV-Ab was due to insufficient PBMC washing and/or cytophilic immunoglobulin (Ig); a particular washing procedure, as well as 24 h peripheral blood mononuclear cells (PBMC) pre-culture, might help in limiting this phenomenon. However, results should be compared with those obtained in cultures containing puromycin especially in infants, where a higher CD16 antigen expression in lymphocytes is likely responsible for increased amounts of cytophilic Ig released in culture supernatants, compared to adults.