An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibodies have great potential, and in particular, efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, Fc" RI, using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with Fc" RI-expressed KU812F cells. Subsequently a combined phage antibody library 6 Â 103 in the size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant Fc" RI and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against Fc" RI. These results suggest that one can obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.