Successful gene silencing by RNA interference requires a potent and specific depletion of the target mRNA. Target candidates must be chosen so that their corresponding short interfering RNAs are likely to be effective against that target and unlikely to accidentally silence other transcripts due to sequence similarity. We show that both effective and unique targets exist in mouse, fruitfly, and worm, and present a new design tool that enables users to make the trade-off between efficacy and uniqueness. The tool lists all targets with partial sequence similarity to the primary target to highlight candidates for negative controls. (C) 2004 Elsevier Inc. All rights reserved.