The assembly of fibronectin (Fn) matrix is a key event in regulating cell adhesion, migration and differentiation. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, we examined Fn fibril assembly from soluble bovine Fn underneath fibroblasts in Xenopus cell cultures and quantitated the resulting matrix by using a selectively cross-reactive antibody. The soluble form of bovine Fn was bath-applied to the cell cultures, and fibroblasts changed the soluble form of Fn into the fibrillar form in a time-dependent manner. Integrin antagonists, Arg-Gly-Asp-Ser peptide and Rhodostomin, inhibited the formation of Fn matrix from soluble Fn. Genistein, cytochalasin D, colchicine, H-7, Ro-31-8220 and forskolin exerted similar inhibitory action. However, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) significantly accelerated the formation of fibrillar Fn. The clustering of integrin and vinculin was enhanced and inhibited by TPA and forskolin, respectively. Following one day's incubation with soluble Fn to preorganize fibrillar Fn matrix, bath application of these drugs caused the degradation of the Fn network, indicating that Fn fibrillogenesis is a dynamic state modulated by cytoskeleton and protein kinases. Aprotinin and leupeptin selectively antagonized the Fn degradative action of forskolin, but not that caused by H-7. These results suggest that the dynamic assembly of Fn matrix is integrin- and cell-dependent and that both PKA and PKC play important roles in regulating the dynamics of Fn matrix. PKC activation enhanced the assembly of Fn, whereas inhibition of PKC caused the disassembly of Fn matrix. On the other hand, activation of PKA inhibited Fn assembly and caused the degradation of Fn matrix mainly via the activation of multiple proteolytic enzymes. Serine and cysteinyl proteases play important roles in Fn disassembly caused by PKA activation, but not that caused by PKC inhibition.