Three tyrosine-to-phenylalanine mutants of ribonuclease A (Y25F, Y92F, and Y97F) are investigated for their enzymatic activities, molecular stabilities, and unfolding/refolding kinetics. These mutants exhibit 80, 90, and 80%, respectively, of the catalytic activity of the wild-type enzyme. Thermal, Gdn.HCl, and pH transition measurements indicate that Y25F and Y97F are less stable than the wild-type protein, whereas the bulk of the thermodynamic and kinetic evidence indicates that Y92F is as stable as the wild-type protein. Differences in molar extinction coefficients indicate that tyrosines 25, 92, and 97 contribute 38, 13, and 39%, respectively, to the absorption difference between the folded and unfolded states, in general agreement with previous studies but possibly indicating the contribution of a fourth tyrosine residue to account for the remaining 10%. Stopped-flow single- and double-jump kinetic experiments were carried out on the wild-type and three mutant proteins. At least one tyrosine residue besides tyrosine 92 contributes to the slow fluorescence-unfolding phase; the likely candidate for this residue is tyrosine 115 which monitors the cis-trans isomerization of the X-Pro114 peptide bond. Tyrosines 25 and 97 are involved in interactions that retard conformational unfolding and accelerate conformational refolding as well as the cis-trans proline isomerization of the slow-refolding phases, presumably by stabilizing the major beta-hairpin structure of RNase A. These interactions may contribute to the strong pH dependence of the folding and unfolding of ribonuclease A. In contrast, tyrosine 92 does not affect the folding and unfolding rates significantly. An improved "box" model of proline isomerization under unfolding conditions was derived from exhaustive fitting of all possible box models. The kinetic data support the identification of Pro93 as the proline whose isomerization distinguishes the slow-refolding species (USII and USI) from the other, faster-refolding species (Uvf, Uf, and Um), implying that Pro93 isomerizes in the slow-refolding reactions USI --> N and IN --> N. Similarly, Pro114 seems to distinguish between the very fast-refolding species Uvf and the fast-refolding species Uf. Lastly, Pro117 seems to distinguish the major slow-refolding species USII from the minor slow-refolding species USI and the medium-refolding species Um from the fast- and very fast-refolding species.