The neural basis of sexual orientation in Drosophila was studied by the production of males with regionally feminized brains. Such flies express the female form of the sex determination gene transformer in a limited number of neurons under the control of GAL4 enhancer trap inserts. This method facilitated the creation of lines with a stable pattern of feminization. In tests of sexual preferences, flies that were feminized in a portion of the antennal lobes or in a subset of the corpora pedunculata (mushroom bodies) courted both males and females. These two brain structures, both of which are involved in olfactory processing, may function in the recognition of sex-specific pheromones, in the control of sex-specific behaviors, or both.