Tumor cell adhesion to extracellular matrix (ECM) and its stabilization are important determinants in metastasis formation, and they are mediated, in part, by integrins and regulated by a variety of protein kinases. Protein tyrosine kinase pp60c-src is found in adhesion-dependent focal adhesion plaques where it may regulate different integrin-mediated signaling cascades. Using human HT-29 colon carcinoma cells stably transfected with pp60c-src--specific antisense oligonucleotides (HT-29AS15), we investigated the role of pp60c-src in integrin-mediated adhesion and its stabilization to ECM components collagen I or IV under static and laminar fluid flow conditions. Under static adhesion conditions transfection of pp60c-src antisense oligonucleotides did not modify adhesive properties. Phosphorylation of focal adhesion kinase (FAK) and paxillin induced by static adhesion to collagen I or IV were similar in HT-29P and HT-29AS15 cells. However, using hydrodynamic conditions in a laminar flow chamber we found a slight reduction in early adhesion events and an even greater difference in adhesion stabilization rates (ASRs). The transfected cells showed a significant reduction in their ability to withstand shear forces and stabilize adhesive bounds. These changes correlated with the cellular expression levels of pp60c-src. Our results suggest that pp60c-src may be involved in stabilization of dynamic HT-29 cell adhesion to ECM components, and this kinase appears to be part of a mechanosensory protein complex during integrin-mediated cell adhesion.