Kinetic analysis, using substrates that consisted entirely of deoxynucleotides with the exception of the single mandatory ribonucleotide at the cleavage site which contained either a 5’-oxy- or 5’-thio-leaving group, demonstrated that the departure of the S-leaving group was not the rate-limiting step of a hammerhead ribozyme-catalyzed reaction [Kuimelis, R. G.; McLaughlin, L. W. J. Am. Chem. Sec. 1995, 117, 11019-11020]. We recently synthesized a natural all-RNA substrate that contains a 5’-thio-leaving group at the cleavage site and performed detailed kinetic analysis. In contrast to the conclusion of Kuimelis and McLaughlin, we found that (i) the attack by the 2’-oxygen at C-17 on the phosphorus atom is the rare-limiting step only for the substrate that contains a 5’-thio group (R11S) and (ii) the departure of the 5’-leaving group is the rate-limiting step for the natural all-RNA substrate (R110) in both enzymatic and nonenzymatic reactions.