Soluble human plasma fibronectin or collagen types I or IV, when preincubated with tissue culture plastic dishes, were effective spreading agents for cultured human keratinocytes and increased spreading in a time and concentration-dependent manner. Spreading on fibronectin, but not on type IV collagen, was inhibited by antifibroneetin; therefore, the contribution of fibronectin to the spreading activity of the natural matrix produced by keratinocytes could not be determined using antifibronectin. Fibroneetin mediated spreading at both high (1.1 mM) and low (0.1 mM) Ca⁺⁺ concentrations, and spreading was not altered by cycloheximide. Insoluble fibronectin deposited by keratinocytes correlated with phagokinetic tracks on particulate gold salts, and added fibronectin, as well as type I collagen and type IV collagen, enhanced motility of keratinocytes. These studies show that production of fibronectin and responsiveness to it are similar in fibroblasts and keratinocytes and demonstrate that fibronectin can act as a matrix factor for keratinocytes.