We explored the possibility of using a genetic approach to inhibit integrin-mediated endothelial cell adhesion and survival. We constructed recombinant adenoviruses (Ads) expressing chimeric proteins consisting of the cytoplasmic and transmembrane domains of integrin beta1 (CH1), beta3 (CH3) or the beta1 transmembrane domain alone (CH2) connected to the extracellular domain of L3T4 placed under the control of the CMV promoter (AdCMV) or the endothelial cell specific Tie-1 promoter (AdTie). All constructs were expressed in a dose- and time-dependent manner with over 90% of cells expressing the constructs within 24 h (AdCMVs) or 72 h (AdTies) after infection. Confluent monolayers of HUVEC infected with AdCMVCH1 or AdCMVCH3 detached from the substrate in a time- and dose-dependent manner with over 95% of the cells being detached 2 days (AdCMVs) or 3 to 4 days (AdTies) after infection. Cell detachment was preceded by the disruption of focal adhesions and reorganization of the actin cytoskeleton and was associated with a reduced ligand-binding activity of beta1, while cell surface density of beta1 integrins remained unchanged. Detached cells failed to re-adhere to different matrix proteins, without, however, any specificity toward beta1 or beta3 integrin-mediated adhesion. Upon detachment, HUVEC rapidly died by apoptosis. These results demonstrate that dominant negative inhibition of integrin function is an effective approach to disrupt endothelial cell adhesion and survival in vitro.