Protein purification often involves the use of denaturing agents for solubilization. During refolding, following removal of the denaturants, the proteins of interest are exposed to proteases present in the expression system. Here the resistance of selected recombinant proteins to three widely used proteolytic enzymes, trypsin (EC 3.4.21.4), proteinase K (EC 3.4.21.14) and endoproteinase Glu-C (EC 3.4.21.19), was investigated during folding and in the folded state. Target proteins and protease mixtures were denatured in 8 mol dm(-3) urea and the proteins were allowed to refold by removal of the urea by dialysis. The proteolytic products were analyzed by sodium dodecyl sulfate-polyacrylamide gels and the protein digestion during folding was compared with the digestion under similar conditions in physiological buffer. Depending on the folding state of the proteins, the proteases had different effects on the substates. During folding, certain recombinant proteins were more efficiently digested by trypsin and, in particular, by proteinase K in comparison with digestion in the folded state, while other protein substrates were more resistant to proteolytic degradation in a denatured or partially denatured state than their folded counterparts. Incubation of most substrate proteins with endoproteinase Glu-C yielded kinetics of digestion that were essentially similar for both partially folded and unfolded substrates. The results reported may be useful for protection of sensitive proteins and in studies of protein folding mechanisms.