Antibodies of the IgG4 subclass, directed against cell surface antigens have received attention as therapeutic molecules due to their poor induction of the complement system. The MHC class II-directed IgG4 antibody 1D09C3 has been explored for the treatment of lymphomas. The mechanism-of-action is still controversial. Apoptosis induction following HLA-DR engagement has been proposed. However, the validity of these results has been questioned by the observation that antibodies may induce formation of cell aggregates and cell death is induced upon dispersion of these aggregates prior to the quantification of cell death by flow cytometry. Here we address the capacity of 1D09C3 to induce apoptosis in vitro, also taking account of the recently reported Fab arm exchange of IgG4 antibodies. 1D09C3 induces formation of tight cellular aggregates that can only be dispersed at the expense of massive cell damage and death. Using dual color fluorescence cross-correlation spectroscopy (FCCS) we demonstrate that also this antibody undergoes Fab arm exchange in the presence of IgG4. FCCS is a powerful technique to investigate the molecular mechanism of Fab arm exchange using minute amounts of reagents. Following exchange, the functionally monovalent 1D09C3 chimeras loose their ability to induce aggregate formation of HLA-DR-positive cells. Neither functionally monovalent nor bivalent 1D09C3 antibodies induce cell death or apoptosis in myeloma target cells, when microscopy instead of flow cytometry is employed as the analytical technique. Our results indicate that the activity of 1D09C3 in vitro may have been a consequence of assay design rather than an ability to induce HLA-DR-dependent cell death.