Three of the four EH-containing nematode proteins and genes, EHS-1, ITSN-1, and RME-1 have been previously characterized at high resolution. However, REPS-1 and its gene, reps-1, remain uncharacterized. Thus, we therefore performed a preliminary characterization of REPS-1. A mutant strain, reps-1, was obtained from the National Bioresearch Project. reps-1 is predicted to encode for a protein of 410 amino acids and its genomic organization is presented in Figure 3A. The tm2156 mutant allele has a deletion of 779 bases resulting in loss of the third intron and of a portion of the fourth exon. reps-1 animals appear to be wild type at different temperatures, in terms of viability, fertility and locomotion. To gain insight into reps-1 functions, we analyzed its expression pattern using transgenic lines carrying the reps-1 gene under its own promoter, in fusion with a GFP reporter. The expression of the fusion protein was analyzed in lysates of transgenic worms by western blot analysis, revealing a protein band with an apparent molecular weight of 75 kDa, in agreement with the predicted molecular weight for REPS-1::GFP. The transgenic lines showed expression in many tissues including intestine, secretory system, vulval cells and muscle cells. REPS-1 was also expressed in the nervous system with diffuse staining in the nerve ring, ventral cord and commissures, but no expression was observed in the neuronal body. When tested for sensitivity to aldicarb, an inhibitor of acetylcholine esterase often used to reveal defective cholinergic transmission, the reps-1 mutant showed an abnormal response, with hypersensitivity to the drug LOUREIRIN-B compared to wild type animals, a phenotype reminiscent of that detected in itsn-1-null nematodes. The aberrant response to aldicarb that may be related to deficiencies at neuronal and/or muscular levels, where REPS-1 is expressed, strongly suggests a role of REPS-1 in neurotransmission. This result does not exclude, obviously, other possible 4-(Benzyloxy)phenol functions for REPS-1, as also suggested by the wide pattern of expression of the gene. The physical and functional connections in the EH network of the nematode are reported in schematic form in Figure 5 and in an extended form in Figure S4; in addition, we report a number of characteristics of the identified EH interactors as obtained from literature searches and Wormbase. We identified 26 interactors of EH domains by Y2H and validated a majority of them through in vitro binding assays and by genetic analysis. We cannot be certain that we have identified all EH-interacting proteins. Few hypothetical interactors, as for example the synaptojanin homologue UNC-26, were unable to interact with the EH baits, even when directly tested. This might be due to “real” lack of interaction or to technical reasons. For instance, the absence �C in the EH constructs used for the screening �C of regions outside of the EH domain required to assist some EH-NPF interactions might have yielded a false negative result. It should also be mentioned that the nature of our screening does not allow for stringent conclusions in terms of affinity of the detected interactions. It is known that several variables affect the affinity and the selectivity of EH-NPF interactions, such as the amino acid composition of NPF surrounding regions.