In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is required to maintain behavioral rhythms under constant conditions. To understand how PDF exerts its influence, we performed time-series immunostainings for the PERIOD protein in normal and pdf mutant flies over 9 d of constant conditions. Without pdf, pacemaker neurons that normally express PDF maintained two markers of rhythms: that of PERIOD nuclear translocation and its protein staining intensity. As a group, however, they displayed a gradual dispersion in their phasing of nuclear translocation. A separate group of non-PDF circadian pacemakers also maintained PERIOD nuclear translocation rhythms without pdf but exhibited altered phase and amplitude of PERIOD staining intensity. Therefore, pdf is not required to maintain circadian protein oscillations under constant conditions; however, it is required to coordinate the phase and amplitude of such rhythms among the diverse pacemakers. These observations begin to outline the hierarchy of circadian pacemaker circuitry in the Drosophila brain.