RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (Ki) toward subtilisin was 2.5 microM.