Injection of the human neurotropic polyomavirus, JCV, into neonatal hamsters causes tumors of glial origin. Previously, a rapidly proliferating cell line, HJC-15, which expresses high levels of the viral T-antigen, had been established from JCV-induced hamster glial tumors. In our analyses of the mechanisms involved in the control of glial cell proliferation in these tumor cells, we have focused our attention on E2F1, a DNA-binding transcription factor which modulates the activity of genes involved in the S-phase of the cell cycle. Here, we report the identification of a novel nucleo-protein complex that forms between select E2F1-binding sites and nuclear proteins from HJC-15 and normal hamster glial cells. In comparison to the previously characterized E2F1 complexes, this complex exhibited distinct mobility, binding and biochemical characteristics. This slower migrating complex also contained several unique Glial E2F1-associated proteins, (GEAP), which have a distinct molecular mass. Of particular, unlike the classical E2F1 whose DNA binding activity is increased during S-phase, the level GEAPs remained constant throughout the cell cycle. GEAPs appeared to confer an increased basal transcriptional activity of promoters containing select E2F1 sites in HJC-15 cells. Interestingly, the increased transcriptional activity modulated by GEAPs in HJC-15 cells was overcome by overexpression of E2F1 in these cells. These data point to the presence of novel members of the E2F family in hamster glial cells with the potential to regulate expression of S-phase specific genes in glial tumors obtained upon intracerebral injection of JCV. The importance of these findings in the pathogenesis of viral-induced tumors and the role of cell cycle regulatory proteins in brain tumor formation is discussed.