In Drosophila, locomotor activity is sexually dimorphic and the brain area controlling this dimorphism has been mapped. The neurons of the pars intercerebralis (PI) have been suggested to participate in such differences between males and females. However, the precise physical nature of the dimorphism, the identity of the PI neurons involved, and the nature of the neuronal signal coding the dimorphism remain unknown. In this study, we used a video-tracking paradigm to characterize further the pattern of locomotor activity in Drosophila. We show that the number of activity/inactivity periods (start/stop bouts) is also sexually dimorphic, and that it can be genetically feminized in males. Moreover, the transplantation of PI neurons from a female, or of feminized PI neurons from a donor male into a receiver wild-type male is sufficient to induce the feminization of locomotor behavior, confirming that this tiny cluster of approximately 10 neurons is directly responsible for the sexual dimorphism in locomotor activity. Finally, feeding males with fluvastatin, a juvenile hormone (JH) inhibitor, also led to start/stop feminization, and this effect is reversible by the simultaneous application of methoprene, a JH analog, suggesting the existence of a neuroendocrine control, by JH, of such behavioral dimorphism.