Previous studies have shown that collagen gel overlay induced selective proteolysis of focal adhesion complex proteins in Madin-Darby canine kidney (MDCK) cells. In this study, we examined whether morphological and biochemical changes were present in cells cultured on collagen gel. We found that focal adhesion complex proteins, including focal adhesion kinase (FAK), talin, paxillin, and p130cas, but not vinculin, were decreased within 1 h when MDCK cells were cultured on collagen gel. Collagen gel-induced selective decrease of focal adhesion proteins was observed in all lines of cells examined, including epithelial, fibroblastic, and cancer cells. Matrigel also induced selective down-regulation of focal adhesion proteins. However, cells cultured on collagen gel- or matrigel-coated dishes did not show any changes of focal adhesion proteins. These data suggest that the physical nature of the gel, i.e. the rigidity, is involved in the expression of focal adhesion proteins. The collagen gel-induced down-regulation of focal adhesion complex proteins was caused by reduction of protein synthesis and activation of proteases such as calpain. Overexpression of a dominant negative mutant of discoidin domain receptor 1 (DDR1) or FAK-related non-kinase (FRNK) did not prevent collagen gel-induced down-regulation of the focal adhesion complex protein, whereas an anti-alpha2beta1 integrin-neutralizing antibody completely blocked it. Taken together, our results indicate that the rigidity of collagen gel controls the expression of focal adhesion complex proteins, which is mediated by alpha2beta1 integrin but not DDR1.