The American horseshoe crab, Limulus polyphemus, excretes nitrogenous waste in the form of highly toxic ammonia across their book gills. The mechanism of this branchial excretion is yet unknown. In another marine arthropod, the green crab Carcinus maenas, a novel ammonia transporter has been discovered and shown to be expressed in the gills. In the current study, two isoforms of this transporter, LpHIAT1α and LpHIAT1β, have been identified in L. polyphemus. Both isoforms have 12 predicted transmembrane regions and share 82.7% of amino acid identity to each other. Significantly higher mRNA expression levels of LpHIAT1α were observed in the peripheral mitochondria-poor region of the gill (PMPA), central mitochondria-rich region of the gill (CMRA), and in the brain compared to the LpHIAT1β isoform. Heterologous expression of LpHIAT1α and LpHIAT1β in Xenopus laevis oocytes resulted in a significantly lower uptake of the radiolabeled ammonia analogue 3H-methylamine compared to water-injected control oocytes, suggesting an excretory function of this transporter. Exposure to elevated environmental ammonia (HEA, 1 mmol l-1) caused an increase in mRNA expression of LpHIAT1β in the ion-conductive ventral gill half. The high mRNA expression of both isoforms in the brain, and the LpHIAT1α- and LpHIAT1β-mediated methylamine excretion suggest that these highly conserved ammonia transporters have an imperative protective function in cellular ammonia detoxification.