The mechanisms by which circadian pacemaker systems transmit timing information to control behavior are largely unknown. Here, we define two critical features of that mechanism in Drosophila. We first describe animals mutant for the pdf neuropeptide gene, which is expressed by most of the candidate pacemakers (LNv neurons). Next, we describe animals in which pdf neurons were selectively ablated. Both sets of animals produced similar behavioral phenotypes. Both sets entrained to light, but both were largely arrhythmic under constant conditions. A minority of each pdf variant exhibited weak to moderate free-running rhythmicity. These results confirm the assignment of LNv neurons as the principal circadian pacemakers controlling daily locomotion in Drosophila. They also implicate PDF as the principal circadian transmitter.