Double-stranded (ds) RNA interference (RNAi) is a recent technological advance that enables researchers to reduce gene expression at the post-transcriptional level. This form of RNA silencing is initiated by dsRNA, expressed in or introduced into a cell of interest, which triggers homology-dependent degradation of the corresponding mRNA. This versatile technique has remarkable promise as a tool for the study of eukaryotic pathogens. Protozoan parasites and pathogenic fungi often resist manipulation using standard molecular genetic approaches. Researchers studying these organisms need flexible molecular tools, particularly to exploit newly sequenced genomes; this review offers a practical guide to establishing RNAi in pathogenic eukaryotes.