The aim of this study was to determine whether anti-p53 antibodies are of clinical significance as a serological marker in the diagnosis and monitoring of malignancies. A total of 1874 serum samples from 591 patients with various types of cancer, esophageal, gastric, colorectal, pancreatic, hepatocellular, breast, and urogenital cancer, and 436 control individuals were analyzed by immunoblot for antibodies against p53. The anti-p53 antibody test was correlated with expression of conventional tumor markers, survival and the clinicopathological features of malignant disease. Anti-p53 antibodies were found in 23.4% (138/591) of the sera of patients with malignant disease (range 11.5-34%). The detection of anti-p53 serum antibodies had a specificity of 100% for malignancy (p<0.0001). The overall sensitivity of measuring established tumor markers was 62.9% (372/591). The elevation of conventional tumor markers and the presence of anti-p53 antibodies in the sera of patients with malignant disease turned out to be an independent variable (p<0.05). Combination of established tumor markers with the anti-p53 antibody test led to an increase in diagnostic sensitivity of 8% (49/591) (p<0.01). Thus, the independence of anti-p53 antibodies from established tumor markers allows the serological detection of additional tumor patients. Kaplan-Meier analysis revealed a trend toward a poorer prognosis in hepatocellular carcinoma and breast cancer patients who were anti-p53 serum positive. In conclusion, testing for anti-p53 antibodies can increase the diagnostic sensitivity when used in combination with measurement of conventional tumor markers. This increase is achieved without a parallel decrease in specificity.