The CD69 antigen is the earliest activation marker expressed on leukocyte surfaces after stimulation and it has been correlated with disease state in a variety of inflammatory and autoimmune diseases. We were interested in the generation of a human monoclonal antibody (mAb) against the CD69 antigen. To do this, mice carrying human Ig transgenes (on an inactivated endogenous immunoglobulin H and Igkappa background) were immunized with rat cells transfected with the human CD69 molecule. From over 2000 hybridoma clones generated in different fusions, we were able to obtain a human monoclonal antibody, hAIM-29, which specifically recognizes human CD69 on the surface of activated-human leukocytes. We demonstrate that the antibody is specific for the human CD69 molecule, as shown by double staining with mouse anti-human CD69 antibodies, ELISA, immunoblot and immunoprecipitation studies. Results of additional experiments show that hAIM-29 activates intracellular calcium influx without Ig cross-linking and enhances phorbol myristate acetate-induced cell proliferation in a manner similar to other mouse anti-CD69 antibodies. This report is the first to describe the isolation and characterization of a novel human mAb, hAIM-29, which may have therapeutic potential in diseases associated with the presence of activated cells expressing CD69 antigen.