Cell-cell adhesion and cell-substrate adhesion are important interactions that modulate intracellular signaling pathways, as well as various cellular events from gene expression to cell locomotion (Juliano and Haskill, 1993). The full response to adhesion seems to involve not only the cross-linking of integrins but also mechanical input through these receptors (Craig and Johnson, 1996; Wang et al., 1993; Ingber, 1993; Chrzanowska-Wodnika and Burridge, 1996; Choquet et al., 1997). To explore this possibility, we have examined the motility and cytoskeletal organization of NRK epithelial cells and 3T3 fibroblasts cultured on substrates having varying mechanical properties (Pelham and Wang, 1997). Flexible, optically clear substrates were prepared by covalently linking type I collagen to polyacrylamide sheets. The flexibility of the substrate was manipulated by maintaining the acrylamide concentration at 10% while varying the bis-acrylamide contents between 0.03% and 0.26%. In this manner, we were able to maintain a constant chemical environment regardless of substrate flexibility. The Young’s Modulus of the substrate, determined by measuring the extent of stretching in response to known applied forces, showed a 13-fold difference between sheets of 0.26% and 0.03% bis-acrylamide. When probed microscopically with a calibrated microneedle, the substrates showed 16-fold difference in compliance (-7.3 x lo-’newtons/pm versus-4.6 X lop8 newtonslym). On more rigid substrates, both NRK epithelial cells and 3T3 fibroblasts were well spread and appeared indistinguishable from those cultured on glass or plastic sur-