Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. Here we show that expression of either the GFP-tagged N-terminal or the C-teminal halves of cortactin inhibits significantly the spreading of COS7 cells on fibronectin. Introducing inactivating point mutation into the SH3 domain of the C-terminal half of cortactin suspends the dominant negative effect of the construct. In addition, a vector-based RNA interference was used to knock-down endogenous level of cortactin in cells. We demonstrate that cortactin deficient cells were not able to spread. These results suggest that cortactin is required for integrin-mediated signalling pathways.