The genetic information contained within the RNA genome of picornaviruses is expressed as a single large open reading frame; processing of the primary translation product begins while translation is still in progress. In rhinoviruses and enteroviruses, two picornavirus genera, the virally encoded proteinase 2A begins the processing cascade, cleaving between the C-terminus of VP1 and its own N-terminus. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2. This enzyme was shown to have an unusual preference at the P1 site; out of eight amino acid substitutions made, only the branched amino acids Val and Ile were not readily accepted. The HRV2 2A was shown to process poorly the HRV89 2A cleavage site and to be unable to cleave at sites which included the P\' region of poliovirus or HRV14. Furthermore, the 2A of HRV89 preferred the cleavage site of HRV2 to its own.