Human papilloma virus type 16 (HPV-16) and herpes simplex virus type 2 (HSV-2) are human viruses implicated in the development of cancer, in particular cervical cancer. The ability of HSV-2 and HPV-16 to transform early passage human cells was examined in this report. For these studies, gingival fibroblasts were utilized. One gingival cell strain was derived from a normal individual (N-16). The second cell strain was derived from hyperplastic gingival tissue of an epileptic individual (R-30) treated with phenytoin, an antiseizure drug. A common side effect of phenytoin is the induction of gingival overgrowth. R-30 cells contained a stable chromosomal translocation between chromosomes 8 and 18 and expressed higher steady state levels of c-myc. HPV- 16 DNA efficiently immortalized R-30 cells but not N-16 cells. R-30 cells cotransfected with HPV-16, and HSV-2 viral DNAs were more aneuploid than R-30 cells transfected with HPV-16 DNA alone. Additionally, R-30 cells cotransfected with both viral DNAs grew better in soft agar than R-30 cells transfected with HPV-16 DNA alone. HSV-2 DNA was detected in transformed cells by polymerase chain reaction. These results suggested R-30 cells were immortalized more efficiently by HPV-16 and further imply that HPV-16 and HSV-2 DNA fragments can cooperate during multistep transformation.