DNA-binding E2F complexes have been identified throughout the mammalian cell cycle, including the transcriptionally inactive complexes with pocket proteins, which occur early in the prereplicative G(1) phase of the cycle, and the transactivating free E2F, which increases in late G(1). Here, a regulatory B-myb promoter site was shown to bind with high affinity to free E2F and to E2F-pocket protein complexes in an indistinguishable way in vitro. In contrast, in vivo footprinting with NIH 3T3 cells demonstrated E2F site occupation specifically in early G(1), when the B-myb promoter is inactive. These observations indicate that a novel mechanism governs E2F-DNA interactions during the cell cycle and emphasize the relevance of E2F site-directed transcriptional repression.