Rad50, an structural maintenance of chromosomes (SMC) protein family member, participates in a variety of cellular processes, including DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiosis. Disruption of Rad50 in mice leads to lethality during early embryogenesis, indicating its essential function in normal proliferating cells. In addition to its ability to form a complex with the DNA double-strand break repair proteins Mre11 and NBS1, Rad50 may interact with other cellular proteins to execute its full range of biological activities. A novel 87-kDa protein named RINT-1 was identified using the C-terminal region of human Rad50 as the bait in a yeast two-hybrid screen. Human RINT-1 shares sequence homology with a novel protein identified in Drosophila melanogaster, including a coiled-coil domain within its N-terminal 150 amino acids, a conserved central domain of about 350 amino acids, and a C-terminal region of 90 amino acids exhibiting 35--38% identity. The conserved central and C-terminal regions of RINT-1 are required for its interaction with Rad50. While Rad50 and RINT-1 are both expressed throughout the cell cycle, RINT-1 specifically binds to Rad50 only during late S and G(2)/M phases, suggesting that RINT-1 may be involved in cell cycle regulation. Consistent with this possibility, MCF-7 cells expressing an N-terminally truncated RINT-1 protein displayed a defective radiation-induced G(2)/M checkpoint. These results suggest that RINT-1 may play a role in the regulation of cell cycle control after DNA damage.