The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130-->Tyr almost completely inhibited enzyme activity at 37 degrees C; activity was, however, partially restored by the following exchanges: Ser-27-->Pro, His-135-->Arg or His-137-->Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130-->Tyr, Phe-130-->Tyr/His-135-->Arg, Phe-130-->Tyr/His-137-->Arg, His-135-->Arg or His-137-->Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli strain BL21(DE3), and the structural stability, as monitored by 8-anilino-I-naphthalenesulphonic acid fluorescence and CD spectrometry, were affected. The thermal transition temperatures for both the activity and the stability of the Phe-130-->Tyr 2A proteinase were reduced by about 17 degrees C compared with the wild-type enzyme. The presence of the additional mutations His-135-->Arg or His-137-->Arg in the Phe-130-->Tyr mutant increased temperature stability by 3 degrees C and 6 degrees C respectively. Thus essential interactions exist within the C-terminal domain of human rhinoviral 2A proteinases which contribute to the overall stability and integrity of the enzyme.