To characterize the reaction catalyzed by a hammer-head ribozyme, the dependence on temperature of the reaction was examined. An Arrhenius plot revealed a transition that indicated a temperature-dependent change in the activation energy at around 25°C. Thermodynamic parameters of the reaction were estimated at 10 and 35°C. The analyses led to the following conclusions. At 25-50°C, the chemical cleavage step (k c l e a v ) was the rate-determining step, and the cleavad fragments dissociated from the ribozyme at a higher rate than the rate of the chemical reaction. When the temperature was below 25°C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate-determining step. Above 50°C, the rate of the reaction decreased because, at such high temperatures, the formation of the Michaelis-Menten complex (duplex formation) was hampered by thermal melting. A conformational change in the ribozyme-substrate complex was not the rate-determining step at any of the temperatures examined.