It has previously been established that human rhinovirus 14 protease 3C binds specifically to the 5\'-noncoding region of the viral RNA. A series of mutants of protease 3C and deletion or point mutants of the 5\'-noncoding region of the viral RNA were analyzed to elucidate the sites of interaction between the protease and the RNA. Amino acids in protease 3C essential for RNA binding were found to be discontinuous in the amino acid sequence, and mutations which destroyed RNA binding did not affect the catalytic (proteolytic) activity of protease 3C. Based on the three-dimensional structure of rhinovirus 14 protease 3C, the RNA binding region is located in an extended area distinct from the catalytic triad. A single stem-loop structure of 27 nucleotides (stem-loop d) in the 5\'-noncoding region was necessary and sufficient to bind protease 3C. Mutagenesis of either the base-paired stem or unpaired loop or bulge regions of stem-loop d suggested that the base-paired stem, but not the loop or bulge, carries important determinants of protease 3C binding. This conclusion is strengthened by the observation that rhinovirus 14 protease 3C bound specifically to the 5\'-noncoding region of poliovirus RNA, and only the base-paired stem of stem-loop d is conserved between poliovirus and rhinovirus RNAs.