Fourteen human lung tumors of various histopathological types were subjected to two-dimensional gel electrophoresis (2-DE). Samples were prepared for 2-DE using a nonenzymatic sample preparation (NESP) technique recently established in our laboratory. Variations in the expression of some polypeptides were observed between tumors of different histopathological types. To this end, high expression of beta-tubulin, heat shock proteins 73 and 90, lamin B, and proliferating cell nuclear antigen (PCNA) were observed in small cell lung carcinomas (SCLC). One polypeptide of unknown identity (35 kDa, pI 5.5) was significantly overexpressed in primary lung adenocarcinomas compared with SCLC, squamous cell lung carcinomas, metastatic lung adenocarcinomas from colon and rectum, and normal tissue. The amino acid composition of this polypeptide is presented. In summary, combining the NESP technique and 2-DE is an effective approach to define tumor-specific markers.