The ability to target ribozymes to specifically cleave viral RNAs in vitro has led to much speculation about their potential therapeutic value as antiviral agents in vivo. To transfer a ribozyme’s potential as an antiviral agent from test tubes to cells and organisms successfully, the characteristics that distinguish these settings must be considered. In vitro, ribozymes and substrate RNAs freely diffuse in solution in test tubes, and trans-cleavage reactions are dependent on a diffusive step. In eukaryotic cells, by contrast, many RNAs do not appear to diffuse freely. Instead, they appear to be highly compartmentalized and actively sorted to specific cellular locations. Such RNA trafficking may result in localization of substrate RNAs in a different compartment than ribozymes, which would effectively reduce substrate RNA availability to ribozymes and therefore limit the effectiveness of ribozymes as gene inhibitors.