Exonuclease 1, a class III member of the RAD2 nuclease family, is a structure-specific nuclease involved in DNA metabolism (replication, repair and recombination). We have identified a homologue to Exonuclease-1 from rice (Oryza sativa L. cv. Nipponbare) and have designated it O. sativa Exonuclease-1 (OsEXO1). The open reading frame of OsEXO1 encodes a predicted product of 836 amino acid residues with a molecular weight of 92 kDa. Two highly conserved nuclease domains (XPG-N and XPG-I) are present in the N-terminal region of the protein. OsEXO1-sGFP fusion protein transiently overexpressed in the onion epidermal cells localized to the nucleus. The transcript of OsEXO1 is highly expressed in meristematic tissues and panicles. Inhibition of cell proliferation by removal of sucrose from the medium or by the addition of cell cycle inhibitors decreased OsEXO1 expression. Functional complementation assays using yeast RAD2 member null mutants demonstrates that OsEXO1 is able to substitute for ScEXO1 and ScRAD27 functions. Yeast two-hybrid analysis shows that OsEXO1 interacted with rice DNA polymerase lambda (OsPol lambda), the 70 kDa subunit b of rice replication protein A (OsRPA70b), and the 32 kDa subunit 1 of rice replication protein A (OsRPA32-1). Irradiation of UV-B induces OsEXO1 expression while hydrogen peroxide treatment represses it. These results suggest that OsEXO1 plays an important role in both cell proliferation and UV-damaged nuclear DNA repair pathway under dark conditions.