Based on the finding that the nuclear DNA of cancerous cells is much more unstable than that of non-cancerous cells, yielding a larger amount of single-stranded DNA by acid hydrolysis, we developed a new method of cancer cell detection in ordinary pathological sections by immunohistochemical staining with anti-single-stranded DNA antiserum after acid hydrolysis. Methylated bovine serum albumin was conjugated with heat-denatured calf thymus DNA and used as the antigen of single-stranded DNA, and white male rabbits were immunized with the antigen to obtain the polyclonal antiserum. Ordinary paraffin-embedded sections were prepared from the formalin-fixed biopsy specimens taken from 482 malignant tumors and 73 benign tumors of human epithelial and non-epithelial origins. Additional 82 biopsy specimens of borderline malignancy were also examined. The sections were immunohistochemically stained with the antiserum after RNase digestion and DNA denaturation by hydrolysis with 2 N HCl at 30 degrees C. The acid hydrolysis for 20-30 min was optimum for the specimens fixed with 10% buffered formalin at room temperature for 16-24 hrs, and all cancerous cells were specifically stained positive, in sharp contrast to the negative stainability of all non-cancerous cells including inflammatory cells. This new method gives us decisive help in making diagnosis of malignancy in daily pathological examination. The possibility of malignancy of borderline lesion was discussed.