The electron paramagnetic resonance (EPR) spin labeling technique has been employed to study the properties and conformation of the thiol protease papain in solution, using (1-oxyl-2,2,5,5-tetramethyl-delta 3-pyrroline-3-methyl) methanethiosulfonate (MTS) as the spin label. The measurements of papain's amidase activity corroborate the EPR results. The major findings are: (i) the motion of the MTS spin label is very sensitive to the active site conformation of papain, which may reflect the location of the pyrroline ring of the spin label near the narrow portion of the active site cleft of papain, and thus there may be intimate interactions between the spin label and its environment; (ii) the active site cleft of papain may have a more open structure at intermediate pH (pH 4.2 to 8.0) than at higher (pH > 8.0) or lower (pH < 4.2) pH, which is consistent with the bell-shape pH curve of the enzyme's amidase activity with the optimum pH at pH 7.00; and (iii) the motion of spin label at the active site of free papain in solution becomes slower upon addition of a denaturant (urea or guanidine hydrochloride), suggesting that the denatured enzyme may have a more closed active site cleft. Urea is more effective than guanidine hydrochloride in denaturing papain at low concentration. However, both urea and guanidine hydrochloride can completely inactivate papain at high concentrations. When an appropriate spin label is selected to label the active site of papain (such as MTS spin label), the EPR spin labeling technique may offer additional insight into the conformation of papain over that obtained by optical methods. These results are discussed in terms of possible studies of biofunctional membranes, opaque assemblies in which a biological molecule is attached to a polymeric membrane.