We have investigated the genetic basis for malignant progression of an human papillomavirus type 16 immortalized human keratinocyte cell line (HPKIA) by somatic cell fusion. In this cell culture model system for human papillomavirus type 16-associated tumorigenesis, early passage cells (HPKIA-nt) are nontumorigenic, whereas gamma-irradiated late passage cells (HPKIA-t) are tumorigenic when tested in nude mice. Fusion of HPKIA-t cells with normal human keratinocytes or with HPKIA-nt gave rise to hybrid clones in which the tumorigenic phenotype is suppressed. This observation is in line with complementation studies conducted for other tumor cell systems and indicates that, also in the case of HPKIA-t cells, the tumorigenic phenotype is recessive. Hybrid clones derived by fusion of HPKIA-t with cells of the cervical carcinoma cell lines C4-I, HeLa, or C33A were also nontumorigenic. However, the tumorigenic phenotype was retained in all of the HPKIA-t x SiHa and HPKIA-t x MS751 hybrids tested. The cervical carcinoma cell lines SiHa and MS751, therefore, cannot complement the gene defect(s) related to tumor growth of HPKIA-t cells. This may suggest that HPKIA-t, SiHa, and MS751 cells share the same defect(s) in regulatory pathway(s) involved in tumor suppression. Differences in the steady state levels of viral E6-E7 transcripts were noted for most of the hybrid cells. The observations of this study underline the potential use of nontumorigenic and tumorigenic HPKIA cells to define genetic differences that may also correlate with tumor progression in vivo.