Hammerhead ribozymes are catalytic RNAs that are being used to inhibit endogenous gene expression to study key components of basic biochemical pathways such as angiogenesis. In addition, these ribozymes have the potential to be used as components of gene therapy protocols for the treatment of disease states. We detail here a set of protocols for the design and testing of hammerhead ribozymes that will efficiently inhibit gene expression both in cell culture and in vivo. © 2002 Elsevier Science (USA). All rights reserved.