We developed an in situ hybridization (ISH) method with a higher sensitivity and less background staining than the generally used catalyzed reporter deposition amplification method of in situ hybridization (CARD-ISH). The characteristics of this method are follows: sections heated in citrate buffer (pH6.0) containing 0.1% Triton X-100 showed the strongest signals, and a well-preserved morphology. The strongest signals were observed when borate buffer of biotinyl-tyramide stock and phosphate buffer of working solution were changed to Tris-HCl buffer. Compared with hematoxylin counter staining, alcian blue counter staining made it easier to identify dot, diffuse, and mixed type signals, respectively. Thus, we were able to clearly detect positive signals in the SiHa cell line with 1-2 copies of the integrated human papilloma virus-16 (HPV-16) gene, and in formalin-fixed, paraffin-embedded cervical lesion specimens for the HPV-16 gene.