Fibronectin (FN) from human plasma, as well as recombinant proteins derived from two of its major domains, the N-terminal fibrin-binding domain (FBD) and the cell-binding domain (CBD), were studied by circular dichroism (CD), Fourier transform infrared (FTIR), and fluorescence spectroscopies. Secondary structure estimations derived from both FTIR spectra and far uv CD indicate significant differences between FBD and CBD proteins. Thus, the CBD 33-kDa protein contains 80% beta-sheet and 20% beta-turns, whereas the three refolded FBD recombinant proteins studied, the 12-, 18.5-, and r31-kDa proteins, as well as the plasma-derived p31-kDa protein, all contain some percentage of aperiodic structure, ranging from 14 to 23% for both 31-kDa proteins, and between 33 and 46% for the smaller 12- and 18.5-kDa proteins. The amount of aperiodic structure increases drastically (from 23 to 67%) in a misfolded recombinant 31 kDa, the scrambled 31 kDa. Both native FN and a chimeric 45-kDa protein, consisting of both the FBD 12-kDa and the CBD 33-kDa proteins, were found to resemble the CBD 33-kDa protein itself, i.e., consisting only of beta-sheet and beta-turns and being free of aperiodic structure. These differences between two types of conformation are corroborated by the differences in the environment and spatial configuration of the aromatic amino acids of these proteins, as evidenced from near uv CD and intrinsic fluorescence spectroscopies. Thus, in the near uv CD the typical CBD proteins, i.e., the 33- and 45-kDa proteins display a maximum at 294 nm, whereas the FBD proteins, except for the scrambled 31-kDa protein, all have a characteristic strong negative peak at 300 nm. Similarly, the lambda max of the emission spectra of the whole FN and the 33- and 45-kDa proteins are characteristically blue-shifted (317-326 nm) and with high normalized intensity, when compared with the relatively red-shifted lambda max (338-340 nm) and low intensities of the folded FBD proteins. The scrambled 31-kDa protein again takes exception, having a relatively high normalized intensity due to misfolding. The two major conclusions from this work, attained by a combination of four spectroscopic methods, are: (a) functionally different FN domains, i.e., the FBD and CBD adopt totally different conformations, with the CBD having an almost exclusive beta structure; (b) the functional specificity of the various domains of FN may depend on subtle differences in the flexibility in their protein backbones, with the FBD being more flexible than the CBD.