BACKGROUND: RalA is a member of the Ras superfamily of small GTPases. The Anti-RalA autoantibodies (s-RalA-Abs) act as tumor markers in various types of cancer and are negatively associated with the p53 autoantibodies (s-p53-Abs). This study aimed to evaluate the relationship between s-RalA-Abs and s-p53-Abs in various types of cancer. METHODS: A total of 1833 cancer patients (esophageal cancer, 172; hepatocellular carcinoma, 91; lung cancer, 269; gastric cancer, 317; colon cancer, 262; breast cancer, 364; and prostate cancer, 358) and 73 healthy subjects were enrolled in the study. The levels of s-RalA-Abs and s-p53-Abs were analyzed using enzyme-linked immunosorbent assay, and the positivity rates and relations between the two autoantibodies were evaluated. The cutoff values for s-RalA abs and s-p53 abs were set as mean + 2 standard deviation and the values higher than the cutoff values were defined as positive. RESULTS: The titers in all cancer types were significantly higher than those in the controls (P < 0.01). The positivity rates for s-RalA-Abs ranged between 11.7 and 21.5%, and those for s-p53-Abs ranged between 12 and 28.5%. A combined assay of the two antibodies revealed positivity rates of 20.9 and 44.2%. In Stage 0/I/II tumors, the positivity rates of the combination of the two antibodies ranged between 21.5 and 42.3%. The two autoantibodies were complementary to each other in the prostate and breast cancers, but independent in other carcinomas. CONCLUSION: The combined use of s-RalA-Abs and s-p53-Abs tended to increase the positivity rate in all cancers, including Stage 0/I/II cancers.