The rat intestinal fatty acid binding protein is an almost all β-sheet protein that encloses a large interior cavity into which the fatty acid ligand binds. The protein contains neither cysteine nor proline. In a previous report, six site-directed mutants were obtained, each having a single cysteine residue [Jiang, N., & Frieden, C., (1993) Biochemistry 32, 11015-11021] either in a turn or pointed into the cavity. In this report, each mutant has been unfolded in denaturant and modified with 5-iodoacetamidofluorescein to introduce a large, bulky, and fluorescent group into the protein at a known position. In all cases, fluorescence changes indicated that the modified protein refolded, and circular dichroism measurements suggested that the refolded protein appeared to be mostly β-sheet. Denaturation curves suggest that for two mutants intermediate structures exist at denaturant concentrations well below the midpoint of the unfolding curve. For each modified, folded protein, one- and two-dimensional 1H NMR spectra were accumulated and compared to the unmodified and wild-type proteins. While the spectra for the modified proteins showed a number of changes in chemical shifts, they were also consistent with folded proteins on the basis of the degree of chemical shift dispersion. Of the six modified mutant proteins, two appear to have the fluorescein group located in the cavity, but only one of these did not bind fatty acid. The remaining modified proteins are capable of ligand binding. In contrast to the modified proteins which contained the fluorescein moiety in the cavity, the fluorescein group in the other modified proteins appears to have been forced to the outside or to the surface. It is concluded that the β-sheet structure and the large internal cavity of the protein allow considerable structural perturbations without disrupting the ability of the protein to fold or affecting the nature of the folded structure.