We have studied the ionic strength-dependent change in conformation of fibronectin, half-molecules of fibronectin produced by reduction and carboxyamidomethylation, and proteolytic fragments. In zone sedimentation through glycerol gradients, intact fibronectin sedimented at 13.5 and 10 S in 0.02 and 0.2 M NaCl, respectively, in agreement with previous studies. Half-molecules sedimented at 11.5 and 7.5 S in the two salt concentrations, demonstrating that the change in conformation occurs independently within each half-molecule. Gelatin-binding plasmic fragments of 165-215 kDa showed a similar large shift in sedimentation coefficient, and one of 60 kDa showed a small shift. We conclude that the change in conformation is effected by short range electrostatic interactions along the strand, rather than by attraction of distant segments of the molecule. Electron microscopy showed that both intact fibronectin and half-molecules exist as extended strands at high ionic strength. At low ionic strength the strands are more strongly curved or bent to produce an irregularly coiled compact structure. No regular points of folding or crossover were seen, suggesting that the compact conformation is produced by increased bending over most or all of the strand.