The central complex is one of the most prominent neuropils in the insect brain. It has been implicated in the control of locomotor activity and is considered as a pre-motor center. Several neuropeptides are expressed in circuits of the central complex, and thus may be modulators of locomotor behavior. Here we have investigated the roles of two different neuropeptides, Drosophila tachykinin (DTK) and short neuropeptide F (sNPF), in aspects of locomotor behavior. In the Drosophila brain, DTK and sNPF are expressed in interneurons innervating the central complex. We have directed RNA interference (RNAi) towards DTK and sNPF specifically in different central complex neurons. We also expressed a temperature-sensitive dominant negative allele of the fly ortholog of dynamin called shibire(ts1), essential in membrane vesicle recycling and endocytosis, to disrupt synaptic transmission in central complex neurons. The spontaneous walking activity of the RNAi- or shibire(ts1)-expressing flies was quantified by video tracking. DTK-deficient flies displayed drastically increased center zone avoidance, suggesting that DTK is involved in the regulation of spatial orientation. In addition, DTK deficiency in other central complex neurons resulted in flies with an increased number of activity-rest bouts. Perturbations in the sNPF circuit indicated that this peptide is involved in the fine regulation of locomotor activity levels. Our findings suggest that the contribution of DTK and sNPF to locomotor behavior is circuit dependent and associated with particular neuronal substrates. Thus, peptidergic pathways in the central complex have specific roles in the fine tuning of locomotor activity of adult Drosophila.