Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.