Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood. We report here that the small GTPase Rap1 regulates the adhesion of a number of cell lines to various extracellular matrix proteins including laminin-5. cAMP also mediates cell adhesion and spreading on laminin-5, a process that is independent of protein kinase A but rather dependent on Epac1, a cAMP-dependent exchange factor for Rap. Interestingly, although both alpha3beta1 and alpha6beta4 mediate adhesion to laminin-5, only alpha3beta1-dependent adhesion is dependent on Rap1. These results provide evidence for a function of the cAMP-Epac-Rap1 pathway in cell adhesion and spreading on different extracellular matrix proteins. They also define different roles for the laminin-binding integrins in regulated cell adhesion and subsequent cell spreading.