The cyclin-dependent kinase inhibitor p21(WAF-1/CIP1/MDA-6) (p21) plays a key role in cell cycle inhibition and apoptosis, and is negatively regulated during cell proliferation. Extracellular matrices can affect cellular proliferation, but their effects on p21 have not been entirely elucidated. Herein, we explore the effects of the matrix glycoprotein fibronectin on p21 expression in human lung carcinoma cells. Our studies show that fibronectin stimulates cell proliferation, and that this effect is associated with suppression of p21 and stimulation of cyclin D1 mRNA and protein levels in human lung non-small lung cell carcinoma cells (H1838). In contrast, the matrix protein collagen type 1 had no effect. The suppression of p21 by fibronectin was blocked by inhibitors of the extracellular signal-regulated kinase pathway (PD98095), and the Rho-kinase pathway (Y-27632). Fibronectin stimulated the phosphorylation of Erk and increased Rho protein expression. To determine the molecular mechanism(s) responsible for the inhibitory effects of fibronectin on p21 expression, transient transfection assays were performed with cells expressing a wild-type human p21 promoter construct. In these cells, fibronectin reduced p21 gene promoter activity. Finally, electrophoresis mobility shift experiments revealed that fibronectin decreased nuclear Sp1 binding activity in the promoter region of the p21 gene promoter, and a Sp1 competing oligonucleotide inhibited the fibronectin response. Taken together, our results suggest that fibronectin stimulates lung cancer carcinoma cell growth by reducing the cyclin-dependent kinase inhibitor p21 and by inducing cyclin D1 gene expression. The reduction of p21 by fibronectin appears to be mediated through Erk and Rho-kinase signaling and DNA-protein interactions at the Sp1 site in the p21 gene promoter. These observations unveil a novel mechanism for p21 gene regulation by fibronectin in lung carcinoma cell growth that represents a potential target for therapy.