Enhanced polymer one-step staining (EPOS) is a novel, highly sensitive one-step immunostaining method. This simple and rapid technique was applied to intra-operative frozen diagnosis. The markers of choice were proliferating cell nuclear antigen (PCNA) and Ki-67 antigen. These cell proliferation markers were both identifiable in fresh frozen sections of the human tonsil in approximately 7 min. The suitable staining sequences are as follows. Frozen sections prepared using 3-aminopropyltrimethoxysilane-coated glass slides are immediately fixed, without air drying, for 15 s in a mixture of 50% formalin and 50% methanol for PCNA, and in 10% formalin for Ki-67 antigen. After a brief rinse in phosphate-buffered saline (PBS), sections are incubated with the EPOS antibody for 3 min, followed by PBS rinse for 1 min. The peroxidase activity is visualized in diaminobenzidine-H2O2 solution containing 10 mmol/L imidazole for 2 min. After a light rinse in tap water, the nuclei are briefly counterstained with 5% methyl green. When necessary, endogenous peroxidase blockage in 1% periodic acid solution for 1 min is added before the EPOS antibody incubation. This procedure is applicable to frozen sections of gastric cancers, malignant lymphomas, and brain, liver and peritoneal lesions in which differential diagnosis between benignancy and malignancy was required.