We have investigated the suitability of proteomics for identification of tumor‐associated antigens. First, we compared the proteomes of nontumorous kidney and renal cell carcinoma (RCC) by two‐dimensional gel electrophoresis (2‐DE) and silver staining. Protein patterns were markedly different (∼800 spots in RCCs versus ∼1400 spots in kidney). 2‐DE immunoblotting revealed five RCC‐specific spots, reproducibly reactive with RCC‐patient but not healthy donor control sera. Two of these antigens were isolated by preparative 2‐DE, and identified by Edman sequencing of tryptic peptides. The first antigen, smooth muscle protein 22‐alpha (SM22‐α), is an actin‐binding protein of unknown function predominantly expressed in smooth muscle cells. In situ hybridization revealed that SM22‐α is not expressed in the malignant cells but in mesenchymal cells of the tumor stroma. The second antigen represents carbonic anhydrase I (CAI), an isoform usually not expressed in kidney. Interestingly, a different isoform (CAXII) has previously been identified by serological expression cloning as an antigen overexpressed in some RCCs. In additional assays, antibodies to recombinant CAI or SM22‐α were detected in sera from 3/11 or 5/11 RCC patients, respectively, whereas sera from 13 healthy individuals did not react. In conclusion, serological proteome analysis may be a new tool for the identification of tumor‐associated antigens.