Human bronchial epithelial (HBE) cells adhere to underlying extracellular matrix (ECM) via integrin-type transmembrane receptors. Integrins link the ECM to the cytoskeleton (CSK), establishing a mechanical continuum by which forces are transmitted between the outside and the inside of the cells. The present study investigates the time course of global and actin CSK stiffness of HBE cells (16HBE14o-) growing on various matrix substrates as a function of culture time until confluence, and the concomitant time course of F-actin and adhesion molecule distribution. Our results showed a progressive increase in actin CSK stiffness from cell seeding to confluence, related to acquisition of highly polymerized cortical and cytosolic F-actin organization and up-regulation of certain matrix ligands, such as β1-, α5-, and αv-integrin subunit expression. Moreover, compared to fibrillar type I collagen, reticular type IV collagen used as matrix substrate, appeared to amplify actin CSK stiffness of HBE confluent cells probably in relation to up-regulation of α3-integrin subunit. Taken together, these results support the concept of a close interaction among actin CSK stiffness, structural actin organization, specific integrin molecule involvement, cell spreading, and extracellular matrix.