Hammerhead ribozymes targeted against two unrelated RNA substrates have been prepared. For each substrate, four ribozymes, differing in their hybridising arm length and composition (DNA or RNA), have been synthesised and kinetically characterised. The presence of DNA in the hybridising arms had little effect on the overall cleavage rate when the cleavage step was rate determining. Shortening each of the hybridising arms of ribozymes from 10 to 6 nucleotides generally resulted in modest changes in rate constants for cleavage of the same 13mer substrate. In one case the presence of long RNA hybridising arms significantly impeded the cleavage reaction. Cleavage rates displayed first order dependence on hydroxide ion concentration at low pHs. At higher pH, some ribozymes deviated from this first order dependence because of a change in the rate-determining step, possibly due to a requirement for a conformation change in the ribozyme-substrate complex prior to cleavage. Ribozyme cleavage was strongly dependent on temperature in the range 5-45 degrees C, with an activation energy for the reaction of approximately 60 kJ mol-1. The ribozymes displayed biphasic dependence on magnesium ion concentration; evidence of strong apparent binding (Kd approximately 10 mM) as well as a looser interaction was observed for all ribozymes.