The E6 and E7 proteins of human papillomavirus (HPV) types 16 and 18 are expressed in cell lines derived from cervical cancers and can immortalize primary human keratinocytes. Since expression of E6/E7 has been shown to induce mitotic defects and karyotype instability in primary human cells, we investigated the effect of these viral oncoproteins on the expression and activity of mitotic regulatory proteins. Primary human keratinocytes immortalized by the entire genome or by only the E6/E7 genes of HPV types 16 and 18 displayed 5- to 20-fold increases in the abundance of p34cdc2, cyclin B and cyclin A when compared with normal parental cells. Results obtained from normal and immortalized cells that were derived from identical single donors were similar to those from mixed donor cultures. Increased protein levels were achieved without corresponding increases in mRNA, indicating alterations in translational and/or post-translational control. The histone H1 kinase activities associated with these regulatory proteins were also elevated, but to a lesser extent than the protein levels. Because p34cdc2, cyclin B and cyclin A regulate the entry into and exit from mitosis, increased expression and activity of these proteins could contribute to the mitotic defects and chromosomal aberrations associated with HPV-induced immortalization.