Monitoring human antibody recognition of tumor antigens could have potential diagnostic and prognostic significance. Serological analysis of recombinant cDNA expression libraries of human cancer has identified a number of immunogenic tumor antigens. To identify colon cancer antigens associated with a cancer-related serum IgG response, serum samples from 74 patients with colon cancer and 75 normal blood donors were screened for antibody reactivity to 77 serologically defined tumor antigens. The following 13 antigens reacted exclusively with sera from the colon cancer patients and not with sera from normal blood donors: p53, MAGEA3, SSX2, NY-ESO-1, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, and LMNA. Serum samples from 34 of 74 (46%) colon cancer patients detected 1 or more of these 13 antigens. Fifty-three of 74 colon cancer patients were of known clinicopathological stage. Analysis of antibody frequency showed that 5 of 7 (71%) stage I colon cancer patients, 4 of 11 (36%) stage II patients, 2 of 14 (14%) stage III patients, and 11 of 21 (52%) stage IV patients had serum IgG antibody that reacted with 1 or more of the 13 antigens. The mRNA expression patterns of 8 of these 13 antigens were altered in cancer. Three of the 13 antigens were cancer/testis antigens (MAGEA3, SSX2, and NY-ESO-1), which are expressed exclusively in normal gametogenic tissues and aberrantly expressed in a broad range of cancer types. Quantitative real-time reverse transcription-PCR showed that the mRNA expression levels of 2 antigens, HDAC5 and Seb4B, were down-regulated in colon cancer, 2 other antigens, KNSL6 and KIAA1416, were up-regulated, and another antigen, NY-CO-45, showed a variable level of mRNA expression in colon cancer. With regard to KNSL6 mRNA expression, only trace levels were detected in 15 different normal tissues with the exception of testis, which showed a high level of KNSL6 mRNA expression. In contrast, 9 of 9 colon cancer specimens showed overexpression of KNSL6 mRNA, ranging from 5 to 44 times the level detected in normal colon tissue, indicating that this antigen could also be a valuable therapeutic target.