The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting α-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the α-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.