Osteopontin (OPN) is a noncollagenous component of bone matrix. It mediates cell attachment and activates signal transduction pathways. In this work, bone cells, cultured from fragments of long bones derived from wild-type and OPN-/- ("knock-out") mice, were exposed to pulsatile fluid flow (PFF) over a 60-min period. The medium was assayed periodically for nitric oxide (NO) and prostaglandin E-2 (PGE(2)) release. OPN+/+ cells exhibited a peak of NO production 5-10 min after the onset of PFF, decreasing to a stable plateau at 15 min; much less NO was produced by the OPN-/- cells. PFF resulted in reduced PGE2 release by both cell types, although the reduction was less for the OPN-/- cells in the 15-30 min window. Both cell types exhibited a similar enhancement of cyclooxygenase2 mRNA levels 60 min after initiation of PFF. These results suggest that bone cells require OPN to respond fully to PFF as assessed by increased NO and reduced PGE synthesis.