The Drosophila tachykinin gene (dtk) produces five different neuropeptides (DTK1-5). These are expressed in about 100 interneurons branching in several neuropils in the Drosophila brain, e. g. the fan-shaped body (FB) of the central complex, a neuropil believed to be a higher center for locomotor control. Previously, DTKs have been shown to modulate locomotor behavior: flies virtually devoid of DTKs (accomplished by RNAi) displayed altered activity in Buridan’s arena, monitoring a 15 min period of walking activity between two opposing landmarks (Winther et al, 2006, Mol. Cell. Neurosci. 31: 399). In the present study we wanted to further explore the impact of DTKs on locomotor behavior. We studied the spontaneous walking activity of the fly, over a period of 7 hours, extracting several parameters from video-tracks using Ethovision software. We found that flies with DTK depletion in all DTK-neurons of the central nervous system (driving expression of the RNAi construct with the pan-neural elav-Gal4) and in a small subset of DTK-neurons (FB 104Y-Gal4) were less active compared to their respective control. Total distance moved by these flies was shorter. Wild-type flies strongly avoid the center of the arena, a phenomena called centrophobism (Martin, 2001, Behav. Process.67: 207). DTK depleted flies (both elav-Gal4;DTK-RNAi and 104Y-Gal4;DTK-RNAi) displayed increased centrophobism. We also found that DTK depleted flies (only in elav-Gal4;DTK-RNAi flies) had a disturbed pattern of activity/inactivity periods in males, but not in females. Periods of activity/inactivity are strictly regulated in Drosophila and this aspect of locomotor activity is sexually dimorphic. In summary, we have found that DTKs act as modulators of several aspects of locomotor activity, and as three specific pairs of DTK-neurons have been identified, we hypothesized that they may be involved in the fine regulation of the level of activity and of centrophobism.