Faithful replication of the entire genome once per cell cycle is essential for maintaining genetic integrity, and the origin of DNA replication is key in this regulation. Unlike that in unicellular organisms, the replication initiation mechanism in mammalian cells is not well understood. We have identified a strong origin of replication at the DBF4 promoter locus, which contains two initiation zones, two origin recognition complex (ORC) binding sites and two DNase I-hypersensitive regions within approximately 1.5 kb. Notably, similar to the Escherichia coli oriC, replication at the DBF4 locus starts from initiation zone I, which contains an ORC-binding site, and progresses in the direction of transcription toward initiation zone II, located approximately 0.4 kb downstream. Replication on the opposite strand from zone II, which contains another ORC-binding site, may be activated or facilitated by replication from zone I. We term this new mammalian replication mode 'asymmetric bidirectional replication'.