The accessibility in human plasma fibronectin of the two free sulfhydryl groups per chain to sulfhydryl reagents 5,5/-dithiobis(2-nitrobenzoic acid) (DTNB) and a maleimide derivative has been examined. For soluble Fibronectin, the free sulfhydryl groups are not accessible to DTNB unless urea or guanidine hydrochloride is added [Smith et al. (1982) J, Biol, Chem. 257, 5831–5838]. Upon binding to polystyrene beads, 0.87 ± 0.05 sulfhydryl group per chain becomes titratable to DTNB. Experiments using fibronectin fragments demonstrate that this newly exposed sulfhydryl group is located in a Type III homologous unit between the DNA-binding and the cell-binding domains. the results suggest that, upon adsorption to solid substrates, plasma fibronectin undergoes a conformational change, thereby exposing one buried sulfhydryl group. These findings have implications regarding the “surface activation” of adhesion activities of fibronectin.