The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) initiates proteolytic processing by cleaving between the C-terminus of VP1 and its own N-terminus. It subsequently cleaves the host protein eIF4GI. HRV2 and HRV14 2A(pro) cleave at IITTA *GPSD and DIKSY *GLGP on their respective polyproteins. The HRV2 2A(pro) cleavage site on eIF4GI is TLSTR *GPPR. We show that HRV2 2A(pro) can self-process at the eIF4GI cleavage sequence whereas HRV14 2A(pro) cannot, due to the presence of the arginine residue at P1. The mutations A104C or A104S in HRV14 2A(pro) restored cleavage when arginine was present at P1, although not to wild-type levels. These experiments define residues which determine substrate recognition in rhinoviral 2A(pro).