The neuropeptides pigment dispersing factor (PDF) and vasoactive intestinal peptide (VIP) are known as key players in the circadian clock system of insects and mammals, respectively. In this study, we report the discovery and characterization of a widely conserved PDF-like neuropeptide precursor pathway in nematodes. Using a combinatorial approach of biochemistry and peptidomics, we have biochemically isolated, identified and characterized three PDF-like neuropeptides in the free-living nematode Caenorhabditis elegans. The two PDF encoding genes, which were designated pdf-1 and pdf-2, display a very strong conservation within the phylum of nematodes. Many of the PDF expressing cells in C. elegans play a role in the control of locomotion and the integration of environmental stimuli, among which light. Our real-time PCR analysis indicates that both PDF genes are consistently expressed during the day and do not affect each other's expression. The transcription of both PDF genes seems to be regulated by atf-2 and ces-2, which encode bZIP transcription factors homologous to Drosophila vrille and par domain protein 1 (Pdp1epsilon), respectively. Together, our data suggest that the PDF neuropeptide pathway, which seems to be conserved throughout the protostomian evolutionary lineage, might be more complex than previously assumed.