Technical Abstract: In Tephritidae sex determination is established by orthologs to the Drosophila melanogaster transformer and transformer-2 genes. In contrast, primary signals for sex determination are different in these species corresponding to the number of X chromosomes (XSE) in Drosophilidae species and to the presence of the Y chromosome in the tephritid species. This paper describes the isolation, expression and function of tra and tra-2 orthologs in the economically relevant tephritidae Anastrepha suspensa, and discusses the possible use of these genes in genetically modified organisms for biologically-based pest management. The Astra and Astra-2 genes are highly conserved in structure, regulation and function with respect to those known from other tephritid species. Sex-specific transcripts for Astra were detected, one in females and three in males, whereas Astra-2 had a single common transcript found in both sexes. To test the function of these genes, Astra and Astra-2 RNAi was injected into A. suspensa embryos from a transgenic strain having a Y-linked DsRed marker integration, allowing XY males to be distinguished from XX phenotypic males. Nearly all XX embryos developed into fully masculinized phenotypic male adults with no apparent female or intersexual morphology. Upon dissection abnormal hypertrophic gonads were revealed in XX pseudomales but not in the XY males. Our findings suggest that Astra and Astra-2 are both necessary for female development, and that the potential exists for producing a male-only population when either gene alone or both genes simultaneously are knocked-down, enhancing the efficiency of control programs such as the sterile insect technique.