Southampton virus (SV) is a human enteric calicivirus with a positive-sense RNA genome which encodes a protease as part of a large precursor polyprotein. Expression vectors based on pRSET were constructed carrying the entire 3C-like viral protease (3Cpro) sequence together with flanking sequences from a region of the viral genome 3'-distal to the putative helicase-encoding region. Expression from these vectors in E. coli resulted in discrete protein products with smaller than expected molecular sizes. This confirmed that an active viral protease was produced in E. coli and that the protease was capable of cleaving the expressed protein at defined sites. Expressed cleavage products surrounding the protease region of the viral polyprotein were separated by SDS-PAGE, transferred to PVDF membranes and subjected to N-terminal sequence analysis. Cleavage occurred at an EG dipeptide at the N terminus of the putative VPg (961E/GKNKG) and also at the protease/polymerase boundary (1280E/GGDKG). The N terminus of the protease was released from the VPg C terminus at an EA dipeptide in the sequence 1099E/APPTL. These studies demonstrate that SV enteric calicivirus encodes a 3C-like protease with a specificity similar to the picornaviral 3C protease and that the SV polyprotein is cleaved into at least six mature products.