We previously investigated the primary and secondary responses and hyperimmunization to the T cell-dependent antigen 2,4-dinitrophenyl keyhole limpet hemocyanin (DNP-KLH) in antigen-free (AF), germ-free (GF) and conventional (CV) mice. Both the absolute and relative numbers of DNP-specific IgG-secreting cells in the spleen of AF mice were considerably higher compared to GF and CV mice, especially after hyperimmunization. In the present study we measured the total and DNP-specific IgG concentration in the sera of these hyperimmunized mice using a sensitive sandwich enzyme-linked immunosorbent assay. With respect to the total IgG concentration before and after hyperimmunization, the AF mice showed an almost 13-fold increase after boosting with the antigen; the GF mice showed an approximately 8-fold increase. A slight but non-significant increase was observed in the CV mice. The total as well as the DNP-specific IgG levels in the AF-immunized mice were 2-fold and 5-fold higher compared to GF and CV mice, respectively. With the use of Surface Plasmon Resonance instrumentation (BIAcore, Pharmacia, Uppsala, Sweden) we obtained mean binding affinities (KA) of the polyclonal samples of the three groups of hyperimmunized mice. IgA and IgM samples displayed low affinity for DNP-lysine. The AF mice displayed the highest KA value among IgG antibodies, followed by GF mice, while CV mice showed a 3-fold lower KA compared to AF mice. These differences were mainly determined by the dissociation rate constant (kdiss), since no significant changes were observed in the association rate constant (kass). Furthermore, the sera of the CV mice have a lower percentage of high-affinity antibodies compared to GF and AF mice. These results suggest that besides a higher overall binding affinity seen in AF mice, and to a lesser extent in GF mice, the relative contribution of high-affinity IgG is greater in AF mice compared to CV mice.