E2F is a family of transcription factors implicated in the regulation of genes required for progression through G1 and entry into the S phase. The transcriptionally active forms of E2F are heterodimers composed of one polypeptide encoded by the E2F gene family and one polypeptide encoded by the DP gene family. The transcriptional activity of E2F/DP heterodimers is influenced by association with the members of the retinoblastoma tumor suppressor protein family (pRb, p107, and p130). Here the intracellular distribution of E2F and DP proteins was investigated in transiently transfected Chinese hamster and human cells. In transfected cells, DP-1 did not accumulate in the nucleus unless it was coexpressed with the heterodimeric partners E2F-1, E2F-2, or E2F-3. Domain mapping experiments showed that regions of E2F-1 and DP-1 that are required for stable association of the two proteins were also required for nuclear localization of DP-1. Unlike E2F-1, -2, and -3, E2F-4 did not accumulate in the nucleus unless it was coexpressed with DP-2, p107 and p130, but not pRb, stimulated nuclear localization of E2F-4, either alone or in combination with DP-2. These results indicate that DP proteins preferentially associate with specific E2F partners, and suggest that the ability of specific E2F/DP heterodimers to localize in the nucleus contributes to the regulation of E2F activity.