Ribonuclease (RNase) A and the more stable glycosylated RNase B differ by a carbohydrate moiety (GlcNAc2Man5-9) attached to Asn34. As previously shown, the first proteolytic cleavage sites to appear on thermal denaturation of both enzymes are in the structural region around Asn34. To discriminate the contribution of the modifying moiety to the stabilization toward thermal unfolding, on the one hand, and proteolytic fragmentation, on the other hand, the carbohydrate chain of RNase B was shortened by treatment with glycosidases to obtain GlcNAc-RNase and (GlcNAc)2Man3 -RNase and extended by binding to concanavalin A or concanavalin A-agarose. The results show a saltatory increase of the thermal unfolding constants and transition temperatures of GlcNAc-RNase in comparison to RNase A, whereas the extension of the modification at Asn34 in the other RNase species does not further increase thermal stability. Therefore, the stability difference between RNase A and RNase B derivatives is attributed to the first carbohydrate unit. In contrast, the rate of proteolysis decreases gradually with increasing volume of the modifying moiety. As concluded from the analysis of the primary cleavage fragments, the main degradation pathway is shifted from the Asn34-Leu35 to the Thr45-Phe46 peptide bond due to increasing shielding effects.