Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.