Tissue culture polystyrene (TCPS) supports good attachment of adherent cells whereas unmodified polystyrene (PS) does not, but the mechanism of this difference is not well characterized. We have compared TCPS and PS for the amounts of vitronectin (Vn) and fibronectin (Fn) which adsorb from the fetal bovine serum (FBS) component of the culture medium. The significance of the amounts of Vn and Fn which adsorbed onto TCPS and PS was determined by reference to the concentration dependence of the cell attachment activity of Vn and Fn when adsorbed onto TCPS and PS, assayed using human vein endothelial cells and BHK-21 fibroblasts. The amount of Vn which adsorbed onto TCPS from medium containing 3-30% (v/v) FBS was supraoptimal for the attachment of endothelial cells and fibroblasts. On PS, the amount of Vn which adsorbed from this medium was less than for TCPS and was suboptimal for cell attachment. Higher levels of Fn adsorbed onto TCPS than to PS, but even the amounts of Fn which adsorbed onto TCPS were suboptimal for cell attachment. We propose that the principal mechanistic difference between TCPS and PS for the initial attachment and spreading of cells is that more Vn adsorbs onto TCPS from the serum component of the culture medium.