The RNA genome of Rice tungro spherical virus (RTSV) is predicted to be expressed as a large polyprotein precursor (Shen et al., Virology 193, 621-630, 1993 ). The polyprotein is processed by at least one virus-encoded protease located adjacent to the C-terminal putative RNA polymerase which shows sequence similarity to viral serine-like proteases. The catalytic activity of this protease was explored using in vitro transcription/translation systems. Besides acting in cis, the protease had activity in trans on precursors containing regions of the 3' half of the polyprotein but did not process a substrate consisting of a precursor of the coat proteins. The substitution mutation of Asp(2735) of the RTSV polyprotein had no effect on proteolysis; however, His(2680), Glu(2717), Cys(2811) and His(2830) proved to be essential for catalytic activity and could constitute the catalytic centre and/or substrate-binding pocket of the RTSV 3C-like protease.