Human polyomaviruses, which include JC virus (JCV) and BK virus (BKV), as well as the simian virus 40 (SV40), have been associated with human tumors and have been shown to be highly tumorigenic in experimental animal models. Although the mechanism by which JCV induces tumorigenesis is not entirely clear, earlier studies point to the involvement of the viral early protein T-antigen which has the ability to bind and inactivate tumor suppressors and cell cycle regulatory proteins, such as the retinoblastoma family proteins and p53. We investigated if the distribution between nucleus and cytoplasm of the transcription factors E2F4 and E2F5 is mediated by pRb2/p130 and if the presence of JCV T-antigen may impair this shuttling by sequestering pRb2/p130. The results showed that E2F4 was prevalently localized in the nucleus of both T-antigen positive and -negative R503 cells independently of the cell cycle phase. E2F5 instead was prevalently localized in the cytoplasmic fraction in G(0)/G(1), S-phase synchronized, and asynchronous R503 and R503 T-Ag positive cells. The presence of T-antigen did not influence the subcellular localization of these transcription factors E2F4 and E2F5, at least in this murine cellular model. Moreover, Small interference RNA experiments directed toward silencing the Rb2/p130 gene demonstrated that pRb2/p130 does not play a predominant role in the nuclear transportation of E2F4 and E2F5.