AIM--To determine the size of a cervical biopsy specimen with human papillomavirus (HPV) infection required to enable in situ hybridisation to be carried out with a guarantee of a reliable result. METHODS--In situ hybridisation was carried out in 142 cervical uterine biopsy specimens classified histologically as low grade and high grade squamous intraepithelial lesions. Epithelial length at the level of the basal membrane was measured by image analysis. The specimens were divided into 10 groups based on epithelial length. RESULTS--Of the biopsy specimens, 61.2% were HPV positive. In specimens with an epithelial length below 5 mm 31.9% were HPV positive; in those between 5 and 9 mm in length 67.5% were HPV positive; and in those greater than 9 mm in length 81.8% were positive for HPV. For low grade squamous intraepithelial lesions (n = 90), 68.4% of specimens with an epithelial length greater than 5 mm were HPV positive. For high grade squamous intraepithelial lesions (n = 52), 86.8% of specimens with an epithelial length greater than 5 mm were HPV positive. CONCLUSIONS--For a diagnosis of HPV infection using in situ hybridisation, the minimum length of epithelium in a cervical biopsy specimen should be 5 mm. For high grade squamous intraepithelial lesions, specimens over 5 mm in length are suitable. For low grade squamous intraepithelial lesions, to minimise the number of false negative results, the ideal minimum length is 10 mm.