We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have demonstrated that proteins produced from these clones possess a proteolytic activity in trans on the cleavage site between the TomRSV movement and coat proteins. By amino acid homology of the TomRSV 3C-related protease with other nepo- and comovirus proteases, His1283, Glu1331 (or Asp1354) and Cys1433 have been predicted to constitute the catalytic triad. Site-directed mutagenesis of His1283 to Asp abolished the TomRSV protease activity, in vitro and in vivo. The cleavage site between the TomRSV movement and coat proteins has been determined to be Q/G, by direct protein sequencing. Previously, His1451 located in the substrate binding pocket of the TomRSV 3C-related protease has been suggested to be involved in the cleavage site specificity. We show that an inactive TomRSV 3C-related protease is obtained after substitution of His1451 with Leu. These results are discussed in light of the possible relation of the TomRSV 3C-related protease to 3C-related proteases of nepo-, como- and potyviruses.