Hammerhead ribozymes may be of great therapeutic importance, since they can, in theory, be developed to cleave any undesired target RNA. In order to design ribozymes which are stable against endogenous RNases, we incorporated 2'-acetamido-2'-deoxynucleosides in the catalytic core of a hammerhead ribozyme. The 2'-acetamido function has, like the hydroxyl group, both proton donor and proton acceptor capacities, which seem to be crucial for efficient catalytic activity. However, the presence of 2'-acetamido-2'-deoxypyrimidine residues in the catalytic core caused a considerable drop in cleavage rate. Replacement of the purine residues led to complete loss of the catalytic activity. Surprisingly, these 2'-modifications showed no beneficial influence on the stability of the ribozymes against RNases present in cell culture supernatant containing 10% fetal calf serum.