1-Oleoyl lysophosphatidic acid (LPA) enhanced binding of 125I-labeled fibronectin by cultured MG-63 osteosarcoma cells and human fibroblasts in monolayer cultures up to threefold over control levels. For osteosarcoma cells, LPA was minimally active at 0.1 ng/mL (0.2 nmol/L) and reached maximal activity at 10 ng/mL (20 nmol/L). Increased binding was evident within 10 minutes of treatment of cycloheximide-treated cells with LPA and was due to an increase in the number of fibronectin binding sites. LPA also increased the binding of a fragment containing the 70-kDa amino-terminal region of fibronectin that is primarily responsible for the reversible binding of fibronectin to matrix assembly sites on cell surfaces. Removal of LPA resulted in prompt return of fibronectin binding to baseline levels. These results indicate that LPA is an important enhancer of fibronectin-rich matrix deposition by cultured cells, and it may be the active component in serum and lipoprotein fractions that is responsible for enhancing fibronectin deposition.