Two new members of the mouse origin recognition complex (ORC) have been cloned that are closely related to Saccharomyces cerevisiae ORC4 and ORC5 as well as to their human homolog. Both MmORC4p and MmORC5p have a putative nucleotide triphosphate binding motif. Transcription of MmORC4 and MmORC5 is not suppressed in mouse NIH3T3 fibroblasts made quiescent by serum starvation. The transcription levels of both ORC genes are constantly high in all phases of the cell cycle. A screen based on the two-hybrid approach suggests that the product of the ORC4 gene interacts with the ORC2, but not with the ORC1 protein. The conservation of structure among members of the ORC4- and ORC5-related family of proteins suggests that these proteins play a key role in the initiation of DNA replication in all eukaryotes.