Cellular aggregation in response to surface antigen signaling is a regulated process important for cell-cell interaction and cell migration. We studied dendritic cell (DC) aggregation in response to major histocompatibility complex (MHC) class II antigen ligation using human monocyte derived mature DCs. Crosslinking of MHC class II antigens by monoclonal antibodies in DCs matured by different stimuli, but not in immature DCs, induced the formation of large and long-lived homotypic cell clusters within 1 hour. These aggregates were completely resistant to mechanical disruption and displayed no signs of increased cell death. Heterotypic conjugate formation of mature DCs with lymphocytes and monocytes following addition of MHC class II antibodies occurred as well. DC aggregation required antigen dimerization, the presence of serum and energy, an intact cytoskeleton, and could not be blocked by EDTA, mannan, and monoclonal antibodies specific for integrins. These data underscore the positive role of MHC class II signaling in mature DCs suggesting the possibility of reverse DC activation in the course of antigen specific interaction with lymphocytes.