Fibroblast-myofibroblast transformation, a critical event for enhanced extracellular matrix deposition, involves formation of an actin stress fiber contractile apparatus that radiates from focal adhesions (FA) in the plasma membrane. Activation of adenylyl cyclase (AC, i.e. increases in cAMP) negatively regulates such transformation. Caveolae and their resident protein caveolins scaffold signaling molecules, including AC isoforms, whereas phosphorylated caveolin-1 (phospho-cav-1) may localize at FA. Here, we used adult rat cardiac fibroblasts to examine distribution and expression of AC, phospho-cav-1, and FA proteins to define mechanisms that link increases in cAMP to caveolin-1 phosphorylation, actin/FA assembly, and fibroblast-myofibroblast transformation. Sucrose density gradient centrifugation, immunoblot, and immunohistochemical analysis revealed that, unlike cav-1, phospho-cav-1 enriches in membrane fractions that express FA proteins and localize at the ends of actin stress fibers. We detected AC in both cav-1 and phospho-cav-1 immunoprecipitates, but FA kinase (FAK), phospho-FAK (FAK Tyr-397), paxillin, and vinculin were detected only in phospho-cav-1 immunoprecipitates. Treatment with the AC activator forskolin or a cAMP analog increased cav-1 phosphorylation but decreased FAK Tyr-397 phosphorylation in a cAMP-dependent protein kinase-dependent manner. These events preceded actin cytoskeletal disruption, an effect that was blocked by small interfering RNA knock-down of cav-1. Inhibition of protein tyrosine phosphatase 1B abrogated cAMP-mediated disruption of actin cytoskeleton, cav-1 phosphorylation, and FAK Tyr-397 dephosphorylation. The data thus define a novel organization of signaling molecules that regulate fibroblasts: scaffolding of AC by phospho-cav-1 at FA sites in a caveolae-free microdomain along with components that mediate inhibition of actin/FA assembly and fibroblast-myofibroblast transformation via increases in cAMP.