Amplification of chromosome 11q13 leads to overexpression of a G1 cyclin gene, cyclin D1 (PRAD-1, CCND-1), in many non-cervical human carcinomas. Homology between cyclin D1 and human papillomavirus oncoprotein E7 binding sites for the retinoblastoma tumor-suppressor protein suggests that human papillomavirus oncoproteins cyclin D1, and other cell cycle regulatory proteins may act through a common mechanism in the pathogenesis of human cervical squamous cell carcinoma. We have examined 48 cases of cervical neoplasia by RNA-mRNA in situ hybridization for human papillomavirus mRNA and cyclin D1 mRNA and by immunohistochemistry for cyclin D1 protein expression. Hybridization demonstrated human papillomavirus RNA in all 48 cases with types 6, 16, or 18 in 2, 26, and 20 cases, respectively. Immunohistochemical detection using anti-cyclin D1 rabbit polyclonal antibody 19 demonstrated appropriate cyclin D1 expression at constitutive low levels in normal squamous epithelium and low-grade intraepithelial lesions. Immunohistochemical staining failed to demonstrate significant protein expression in any of the high-grade or invasive lesions. In contrast to the immunohistochemical results, in situ hybridization demonstrated cyclin D1 mRNA overexpression in three of five cases of low-grade squamous intraepithelial lesion, one of eight cases of high-grade squamous intraepithelial lesion, 14 of 18 cases of invasive squamous cell carcinoma, two of five cases of adenocarcinoma in situ, one of seven cases of invasive adenocarcinoma, and two of five cases of small cell undifferentiated carcinoma. Transcriptional activation of cyclin D1 can occur in vivo in human papillomavirus-associated invasive cervical carcinoma, but it does not seem to result in a steady state, increased level of cyclin D1 protein expression. These data indicate a limited role for cyclin D1 protein in the pathogenesis of human papillomavirus-associated invasive cervical squamous carcinoma. They support a model in which human papillomavirus proteins can circumvent cellular requirements for cyclin D1 in human cervical neoplasia.