To develop useful early and/or differential diagnostic markers for pulmonary adenocarcinomas, we generated monoclonal antibodies using A549 cells derived from pulmonary adenocarcinomas as an immunogen. Hybridoma supernatants were immunohistochemically screened for antibody production by AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice by limiting dilutions. From a group of obtained antibodies, an antibody designated as KU-Lu-3 showed cytoplasmic staining. The antigen recognized by KU-Lu-3 was detected by modified two-dimensional immunoblotting, and was determined to be the receptor of activated C kinase 1 (RACK1). To evaluate the utility of KU-Lu-3, we immunohistochemically studied 184 cases of pulmonary carcinoma and paired normal lung tissues, using formalin-fixed and paraffin-embedded tissue microarray sections. The expression was significantly high and frequent in adenocarcinomas but was barely detected in a few squamous cell carcinomas and large cell carcinomas (p<0.0001). Moreover, RACK1 expression was also significantly associated with the pathological stage, tumor size and lymph node status of adenocarcinoma patients, but not with tumor differentiation, or patient age and gender. These results suggest that RACK1 may be a novel differential diagnostic marker for pulmonary adenocarcinomas.