Acid-base regulation is vital for animals and while the inorganic carbon system largely determines body fluid pH, another potentially valuable acid-base pair is ammonia (NH4+/NH3). This study focuses on the American horseshoe crab (Limulus polyphemus), a phylogenetically ancient marine chelicerate with no published studies on its acid-base physiology. Physiological and molecular analyses indicate that Na+/K+-ATPase, Rhesus-protein (Rh), and carbonic anhydrase (CA) are involved in acid-base homeostasis and/or ammonia regulation. This likely occurs in the book gills, which consist of ultrastructurally distinct regions. The ventral half-lamella is ion-leaky and displayed high Rh-protein, cytoplasmic CA, and hyperpolarization-activated cyclic nucleotide-gated K+ channel mRNA expression levels, suggesting a specialization in facilitated CO2 and/or ammonia diffusion compared to the dorsal half-lamella. During hypercapnia acclimation, hemolymph acid-base status exhibited a compensated respiratory acidosis accompanied with signs of metabolic depression. Ammonia influx associated with high environmental ammonia acclimation was successfully counteracted, but induced modifications in acid-base homeostasis. / October 2016