In order to examine the effects of deoxyribonucleotide substitutions in the substrate strand, several chimeric DNA/RNA substrates for a hammerhead ribozyme were chemically synthesized. Measurements of kinetic parameters revealed that a chimeric DNA/RNA substrate, that contained GUC at the cleavage site as ribonucleotides, was cleaved by an all-RNA ribozyme with a threefold higher kcat than that of the wild-type (wt) reaction. Moreover, this chimeric substrate was also cleaved by a DNA-armed ribozyme that has a higher kcat than the all-RNA ribozyme [Shimayama et al., Nucleic Acids Res. 21 (1993) 2605-2611], with a fourfold higher kcat than that of the wt reaction. Km was increased stepwise by 60-fold per substitutions of the strand of stems I and III by deoxyribonucleotides. These observations demonstrate that although substitutions by deoxyribonucleotides in stems I and III decrease the affinity of substrate and ribozyme, rates of chemical cleavage are actually increased, instead of being decreased, with substitutions by deoxyribonucleotides either on the substrate side or on the ribozyme side or even on both in our system.