The specific requirements for divalent cations in the integrin-dependent adhesion and deadhesion of human dermal fibroblasts and human epidermal keratinocytes to various extracellular matrix proteins have been studied in vitro. The adhesion of both cell types to collagen type I and to laminin was enhanced by Mg2+ in a concentration-dependent manner, while Ca2+ dose-dependently antagonized this effect, thus promoting deadhesion. The cation-dependent conversion between adhesion and deadhesion occurred already at 2 to 10 min after addition of the alternate cation and was almost completed at 20 min. Interestingly, Ca2+ could not reverse the Mg2+-enhaneed adhesion of both cell types to fibronectin. Inhibition studies with function-blocking antibodies directed against distinct β1 integrins showed that the Mg2+-enhanced fibroblast adhesion to collagen type I was mediated by the α1β1 and the α2β1 integrins, whereas keratinocyte adhesion to collagen type I was mediated by the α2β1 integrin. Both cell types utilized the α2β1 and the α6β1 integrins for Mg2+-dependent adhesion to laminin and the α5β1 integrin for the adhesion to fibronectin. Integrin expression at the cell surface was not altered, indicating that divalent cation-dependent conformational changes of β1 integrins most likely regulate their functional activity.