Technical Abstract: Engineering sterile traits in plants through the tissue-specific expression of a cytotoxic gene provides an effective way for containing transgene flow; however, the microbial origin of cytotoxic genes has raised concerns. In an attempt to develop a safe alternative, we have chosen the meiosis-critical genes AHP2, RAD51C and SWITCH1 (SWI) as silencing targets for testing the feasibility of engineering sterility in plants using RNAi. Our results demonstrated that silencing of each of the three genes via hairpin RNA constructs driven by the CaMV35S promoter (termed AHPi, RAD51Ci and SWIi lines) yielded a proportion of lines bearing a very similar partially sterile phenotype in which less than 50% of pollen was viable. In addition, a completely sterile phenotype was also found in RAD51Ci and SWIi lines, but not in the AHPi population. Plants with this sterile phenotype either produced normal-looking but non-viable pollen in the case of RAD51Ci lines or displayed a complete absence of pollen in the case of SWIi lines, which suggests that RAD51C and SWI function at early but distinct stages of meiosis. An analysis of the sterile phenotype in SWI-silenced lines indicated that the level of SWI-specific siRNA did not vary in leaf or floral tissues on the basis of whether the line displayed a fertile or sterile phenotype. Instead, sterility was found to be closely related to a substantial reduction in the level of targeted gene transcript in floral tissues. The sterile phenotype observed in silenced lines was further examined through the use of reciprocal crosses, where only female gametes were found to be capable of fertilization when crossed with wt plants. This work demonstrates that generating sterility through the silencing of key genes involved in the regulation of meiosis is indeed feasible and its advantages and potential applications for transgene containment are discussed.