Studies have been carried out on the adsorption properties of plasma fibronectin (FN) on hydrophobic and hydrophilic surfaces. The hydrophobic and hydrophilic surfaces used were nonwettable bacteriological dishes and wettable tissue culture dishes, respectively. At low plasma FN concentrations, there was more plasma FN binding to hydrophobic surfaces than to hydrophilic surfaces. Under these conditions, the binding of antifibronectin to adsorbed fibronectin was much higher on the hydrophilic surfaces than the hydrophobic ones suggesting that adsorption of plasma FN on the two surfaces occurred in different conformations. In the presence of small amounts of serum albumin, however, antibody binding to plasma FN adsorbed on hydrophobic surfaces increased markedly indicating that a more favorable conformation had been attained. The adsorption of plasma FN onto both hydrophobic and hydrophilic surfaces from serum-containing solutions was found to depend on the serum concentration. At low serum concentrations in the incubations (up to 0.1%), there was increased adsorption of plasma FN with increasing serum concentrations. The highest concentration of adsorbed fibronectin was about 12 ng/cm2 and was sufficient to promote complete cell spreading. Above 1.0% serum there was a marked decrease in fibronectin adsorption and at 10% serum very little adsorption occurred. This indicated that at high serum concentrations other serum proteins were able to compete with fibronectin for surface adsorption sites. This was ccrfirmed by determining adsorption isotherms for fibro1:ectin in the presence of 0.5 and 10% fibronectin-depleted serum. In long term cell spreading experiments (1 h to 2 days), cell spreading eventually occurred on hydrophilic surfaces but not on hydrophobic ones in the presence of 10% serum. This could not be accounted for by exchange on the surfaces of fibronectin for adsorbed serum proteins in the absence of the cells. In the presence of the cells, however, there was an increase in fibronectin associated with the hydrophilic surfaces and a decrease in fibronectin associated with hydrophobic surfaces that occurred concomitantly with increased or decreased cell spreading. This result suggested the possibility that in long term cultures, cells deposit endogenous spreading factors on top of or in place of the adsorbed non-fibronectin serum proteins.