Hammerhead ribozymes are catalytic RNA molecules that specifically cleave a target RNA molecule. Herein, we report the design, synthesis, and in vitro analysis of a hammerhead ribozyme targeted to the infectious bronchitis virus (IBV) nucleocapsid mRNA. At a concentration of 0.5 or 10 microM, the ribozyme, designated IBV-N-Rz, effectively cleaved target RNAs in trans (37 C, 10 mM MgCl2, 50 mM Tris). Cleavage products were visualized by agarose gel analysis. The time course of the ribozyme reaction was monitored by agarose gel analysis and relative quantitative reverse transcription-polymerase chain reaction. The amount of target RNA continually declined over a 5-hr period, indicating that the ribozyme was truly catalytic. Although stability and delivery problems must be overcome, a hammerhead ribozyme targeted to the IBV nucleocapsid mRNA most likely has antiviral activity and may be an effective therapeutic/prophylactic reagent in the future.