In euryhaline fishes like the killifish (Fundulus heteroclitus) that experience daily fluctuations in environmental salinity, endothelin 1 (EDN1) may be an important regulator molecule necessary to maintain ion homeostasis. The purpose of this study was to determine if EDN1 and the endothelin converting enzyme (ECE1; the enzyme necessary for cleaving the precursor proendothelin-1 to EDN1) are present in the killifish, to determine if environmental salinity regulates their expression, and to examine the phylogenetic relationships among the EDNs and among the ECEs. We sequenced killifish gill cDNA for two EDN1 orthologues, EDN1A and EDN1B, and also sequenced a portion of ECE1 cDNA. EDN1A and ECE1 mRNA are expressed ubiquitously in the killifish while EDN1B mRNA has little expression in the killifish opercular epithelium or gill. Using in situ hybridization and immunohistochemistry, EDN1 was localized to large round cells adjacent to the mitochondrion-rich cells of the killifish gill, and to lamellar pillar cells. In the gill, EDN1A and EDN1B mRNA levels did not differ with acute (<24 h) or chronic (30 days) acclimation to seawater (SW); however, EDN1B levels increased threefold post SW to freshwater (FW) transfer, and ECE1 mRNA levels significantly increased twofold over this period. ECE1 mRNA levels also increased sixfold over 24 h post FW to SW transfer. Chronic exposure to SW or FW had little effect on ECE1 mRNA levels. Based upon our cellular localization studies, we modeled EDN1 expression in the fish gill and conclude that it is positioned to act as a paracrine regulator of gill functions in euryhaline fishes. It also may function as an autocrine on pillar cells, where it is hypothesized to regulate local blood flow in the lamellae. From our phylogenetic analyses, ECE is predicted to have an ancient origin and may be a generalist endoprotease in non-vertebrate organisms, while EDNs are vertebrate-specific peptides and may be key characters in vertebrate evolution.