Picornaviruses, such as polio, translate their entire genome as a single polyprotein which must be proteolytically processed to produce the mature viral proteins. A majority of these cleavages are catalyzed by the virus-encoded cysteine proteinase, 3C. We report here the design and synthesis of a series of oligopeptide substrates, based upon native 3C cleavage sites, for an HPLC assay of poliovirus 3C proteinase activity. A similar series of peptides based upon human rhinovirus 3C cleavage sites was also examined. The enzyme shows a marked preference for those peptides with a proline in the P\'2 position. A quenched fluorescent substrate suitable for continuous assay of 3C proteinase activity was also synthesized. Both the HPLC assay and the fluorescence assay were used to evaluate a number of potential 3C proteinase inhibitors.