Osteopontin is a secreted phosphoprotein involved in cellular proliferation and associated with tumor progression. Although an intracellular form of osteopontin has been described, its function remains unknown. In this study a novel nuclear location for intracellular osteopontin and a correlation with cell division were demonstrated. Osteopontin distinctly localized to the nucleus in a subset of transiently transfected human embryonic kidney 293 cells. Immunoblotting confirmed the nuclear location of native osteopontin and results from immunofluorescence studies suggested an association between nuclear osteopontin and cell cycle progression. Flow cytometry revealed that nuclear and cellular osteopontin content rose significantly during S and G₂/M phase, respectively. Treatment of cells with the DNA polymerase inhibitor aphidicolin prevented cell cycling and greatly reduced cellular osteopontin content. The intracellular location of osteopontin coincided with Polo-like kinase-1, a member of the Polo-like kinases, which in part through their regulation of centrosome-related events, are integral to successful cellular mitosis. Osteopontin and Polo-like kinase-1 were co-immunoprecipitated from nuclear, but not cystoslic extracts, demonstrating an interaction that is limited to the nucleus, presumably during mitosis. Deletion of the C-terminus of osteopontin militated against nuclear localization and Polo-like kinase-1 interaction. Elevated expression of osteopontin was also associated with an increase in the number of multinucleate 293 cells, whereas transfection of the C-terminal deleted osteopontin decreased the percentage of multinucleate cells below basal levels. These findings implicate intranuclear osteopontin as a participant in the process of cell duplication.