The dependence of epithelial cell spreading on fibronectin-containing media has been investigated, using purified human plasma fibronectin, and two in vitro assays: (1) epidermal outgrowths from measured pieces of mouse ear skin, and (2) spreading of dissociated cells from trypsinized guinea pig epidermis. Using these systems, appreciable spread occurred in media containing whole plasma and plasma deficient in fibronectin but no significant spread occurred in media enriched only in fibronectin. Returning fibronectin to deficient plasma did not enhance or reduce optimal cell spreading. These studies suggest that the factor(s) in human plasma which supports epithelial cell spreading in vitro is not fibronectin and that the chemistry of epithelial and fibroblast cell spreading is different.