Osteopontin (OPN) is a sialated phosphoprotein found in tissues and secreted into body fluids. It is an integrin ligand with pleiotropic functions as an extracellular matrix protein in mineralized tissues and a cytokine that is active in cell signaling (A. B. Tuck, C. Hota, S. M. Wilson, and A. F. Chambers, Oncogene 22:1198-1205, 2003). To determine whether OPN may be important in mucosal defense against viral pathogens, we evaluated the OPN response to rotavirus infection and the extent of diarrhea manifested by infected opn null mutant (opn-/-) mice. Reverse transcription-PCR, Northern and Western blots, and immunohistochemical studies of the HT-29 intestinal epithelial cell line and murine intestine were used to evaluate OPN mRNA and product. Intestinal closed loops and diarrheal observations determined disease severity and duration. OPN mRNA levels increased after infection of HT-29 cells, peaking in 4 to 6 h. Infected cultures contained 925 microg of OPN/ml, while for controls the levels were below detection (50 microg/ml). Infection increased OPN mRNA levels in intestinal tissue between 2 and 24 h postinoculation and increased OPN protein in intestinal fluid. The cellular localization of OPN was supranuclear and apical, and responding cells were diffusely distributed on the villus surface. Three days after infection, closed intestinal loops from opn-/- mice contained more fluid than loops from controls, although secretion levels at the onset of illness were similar. Null mutant mice experienced more intense and prolonged diarrhea than controls. Rotavirus infection of intestinal epithelial cells and murine intestine caused marked increases in OPN mRNA levels and secreted OPN protein. OPN-deficient mice suffered prolonged disease.