Anti-idiotypic vaccination against HIV infection aims at inducing an anti-gp120 immune response through anti-CD4 Abs mimicking epitopes of the gp120 molecule. The mAb IOT4a induces anti-gp120 Abs in rabbits. This study investigates the presence of human serum Abs cross-reacting with anti-CD4 mAbs and gp120 during HIV infection and after parenteral vaccination with IOT4a. Ten HIV-infected volunteers without immunodeficiency were inoculated s.c. with 0.6, 1.2, or 2.4 mg IOT4a. Six booster injections followed until day 35. Sera from study patients, 80 HIV-positive, and 43 seronegative controls were examined by a panel of assays, including an ELISA using competition against biotinylated recombinant CD4, flow cytometry assaying inhibition of anti-CD4 mAbs and biotinylated gp120 binding to CD4-positive lymphocytes, and an IOT4a immunoblot. After vaccination, an increase in competitive binding activity, which was quantitative in ELISA and flow cytometry, was observed. In the ELISA, competition against biotin-CD4 was quantitatively quenched by preincubating sera with r-gp120 or anti-CD4 mAbs such as IOT4a and Leu3a. Naive sera or sera from blood donors had no such effect in the assays employed, while 5/80 HIV sera showed binding qualities similar to vaccinees. These results suggest that 1) CD4 internal-image Abs emerge in a small proportion of HIV-positive individuals, and 2) parenteral vaccination with mAb IOT4a can induce a gp120 cross-reacting immune response that inhibits gp120 binding to CD4.