Abstract 1. 1. Ribonuclease A and two modified forms, ribonuclease S and S-protein, were exposed to a variety of denaturing agents, i.e. , acid, heat urea, dioxane, dodecyl sulfate and cetyldimethylethylammonium bromide. (a) The observed order of stability was: ribonuclease A > ribonuclease S > S-protein. (b) Reversible changes of ribonuclease S in 8 M urea were by a 2-stage process: ribonuclease S → I S-protein → II extensive modification of the S-protein core, and Stage I was the rate-limiting step at 27°. (c) Dioxane (40%) at neutral pH produced reversible changes in ribonuclease S and S-protein, but denaturation of ribonuclease A occurred only below pH 4. (d) Actions of the cationic detergent cetyldimethylaammonium bromide on ribonuclease are complex but clearly involve a disulfide interchange and additional conformational changes from bound detergent. 2. 2. Fluoremetric titration curves showed marked differences for ribonuclease A, ribonuclease S and S-protein that are compatible with an expansion of the protein by electrostatic repulsive forces. 3. 3. Reduction of the disulfide bonds of denatured ribonuclease gave largeincreases in fluorescence. These results suggest that some hydrophobic regions persist as long as the disulfide bonds are intact. 4. 4. Perturbation of the fluorescence of native ribonuclease A by dioxane indicated that most, if not all, fluorescence came from the normal (exposed) tyrosyl residues.