Branchial ammonia transport in freshwater teleosts is not well understood. Most studies conclude that NH₃ diffuses out of the gill and becomes protonated to NH₄⁺ in an acidified gill boundary layer. Rhesus (Rh) proteins are new members of the ammonia transporter superfamily and rainbow trout possess genes encoding for Rh30-like1 and Rhcg2. We identified seven additional full-length trout Rh cDNA sequences: one Rhag and two each of Rhbg, Rhcg1 and Rh30-like. The mRNA expression of Rhbg, Rhcg1, and Rhcg2 was examined in trout tissues (blood, brain, eye, gill, heart, intestine, kidney, liver, muscle, skin, spleen) exposed to high external ammonia (HEA; 1.5 mmolL^-1 NH₄HCO₃, pH 7.95, 15°C). Rhbg was expressed in all tissues, Rhcg1 was expressed in brain, gill, liver and skin, and Rhcg2 was expressed in gill and skin. Brain Rhbg and Rhcg1 were downregulated, blood Rh30-like and Rhag were downregulated, and skin Rhbg and Rhcg2 were upregulated with HEA. After an initial uptake of ammonia into the fish during HEA, excretion was re-established, coinciding with upregulations of gill Rh mRNA in the pavement cell fraction: Rhcg2 at 12 and 48 hours, and Rhbg at 48 hours. NHE2 expression remained unchanged, but upregulated H⁺-ATPase (V-type, B-subunit) and downregulated carbonic anhydrase (CA2) expression and activity were noted in the gill and again expression changes occurred in pavement cells, and not in mitochondria-rich cells. Together, these results indicate Rh glycoprotein involvement in ammonia transport and excretion in the rainbow trout while underscoring the significance of gill boundary layer acidification by H⁺-ATPase.