Alignment of picornavirus proteinase/polymerase sequences reveals this family evolved into five 'supergroups'. Interestingly, the nature of the 2A region of the picornavirus polyprotein is highly correlated with this phylogeny. Viruses within supergroup 4, the , have complex 2A regions with many viruses encoding multiple 2A sequences. In vitro transcription/translation analyses of a synthetic polyprotein comprising green fluorescent protein (GFP) linked to β-glucuronidase (GUS) via individual 2As showed two main phenotypes: highly active 2A sequences-similar to foot-and-mouth disease virus 2A-and, surprisingly, a novel phenotype of some 2A sequences which apparently terminate translation at the C-terminus of 2A without detectable re-initiation of downstream sequences (GUS). Probing databases with the short sequences between 2As did not reveal any potential 'accessory' functions. The novel, highly active, 2A-like sequences we identified substantially expand the toolbox for biomedical/biotechnological co-expression applications.