RNA interference (RNAi) is a posttranscriptional gene silencing method that is triggered by double-stranded RNA (dsRNA). RNAi is used to inactivate genes of interest and provides a genetic tool for loss-of-function studies in a variety of organisms. I have used this method to reveal the physiological roles of a number of endogenous proteins involved in mitochondrial DNA metabolism in Schneider cells, including the mitochondrial single-stranded DNA-binding protein. Here, I present experimental schemes of selective suppression of endogenous gene expression using RNAi in Drosophila Schneider S2 cells. With this method, the function of exogenous wild-type or mutant genes can be evaluated.