Sera from cancer patients specifically suppressed phosphofructokinase (fructose-6-phosphate kinase [PFK], EC 2.7.1.11), a rate-limiting enzyme in the glycolysis pathway. Among 418 cancerous sera, 68.7% evidence suppression; there was no organ specificity. Among 42 sera from early gastric cancer patients, 29 (69.0%) were positive, as were advanced gastric cancer, 14/19 (73.3%) pancreas cancer, and 75/101 (74.3%) lung cancer sera. In contrast 6/50 (12.0%) sera from patients with gastroduodenal ulcer, 3/23 (13.0%) with myoma uteri, and 0/6 with lung tuberculosis were positive. Patients with diabetes mellitus and those receiving steroid hormone therapy showed strong positive suppression. Comparative studies using other tumor markers (immunosuppressive acid protein, carcinoembryonic antigen, alpha-fetoprotein, beta 2-microglobulin, and ferritin) and the same sera used from PFK assay showed that the PFK method was two to three times more sensitive. Sephadex G-200 column chromatography revealed that the PFK-suppressive activity was retained in the postalbumin fraction. The PFK method may represent a promising new cancer screening method.