Periviscerokinins (PVKs) and pyrokinins (PKs) are neuropeptides known in several arthropod species. Sequence homology of these peptides with the molluscan small cardioactive peptides reveals that the occurrence of PVKs and PKs is not restricted to arthropods. Our study focuses on the biochemical and immunocytochemical identification of neuropeptides with sequence homology to PVKs and PKs in the central and peripheral nervous system of the earthworm Eisenia fetida. By means of affinity chromatography, nanoflow liquid chromatography, and high accuracy mass spectrometry, six peptides, SPFPR(L/I)amide, APFPR(L/I)amide, SPLPR(L/I)amide, SFVR(L/I)amide, AFVR(L/I)amide, and SPAFVR(L/I)amide, were identified in the central nervous system with the common −XR(L/I)amide C-terminal sequence. The exact anatomical position of 13 labeled XR(I/L)amide expressing neuron groups and numerous peptide-containing fibers were determined by means of immunocytochemistry and confocal laser scanning microscopy in whole-mount preparations of ventral nerve cord ganglia. The majority of the stained neurons were interneurons with processes joining the distinct fine-fibered polysegmental tracts in the central neuropil. Some stained fibers were seen running in each segmental nerve that innervated metanephridia and body wall. Distinct groups of neurosecretory cells characterized by small round soma and short processes were also identified. Based on immunoelectron microscopy six different types of labeled cells were described showing morphological heterogeneity of earthworm peptides containing elements. Our findings confirm that the sequence of the identified earthworm neuropeptides homologous to the insect PVKs and PKs suggesting that these peptides are phylogenetically conservative molecules and are expressed in sister-groups of animals such as annelids, mollusks, and insects. J. Comp. Neurol. 514:415–432, 2009. © 2009 Wiley-Liss, Inc.