Lampreys are the most basal vertebrates with an osmoregulatory strategy. Previous research has established that salinity tolerance of sea lamprey increases dramatically during metamorphosis, but underlying changes in the gut have not been examined. In the present work, we examined changes in intestinal function during metamorphosis and seawater exposure of sea lamprey (Petromyzon marinus). Fully metamorphosed sea lamprey had 100 % survival after direct exposure to 35 ppt SW and only slight elevations in plasma chloride (Cl-) levels. Drinking rates of sea lamprey juveniles in seawater were 26-fold higher than juveniles in FW. Na+/K+-ATPase (NKA) activity in the anterior and posterior intestine increased 12- and 3-fold respectively during metamorphosis, whereas esophageal NKA activity was lower than in the intestine and did not change with development. Acclimation to SW significantly enhanced NKA activity in the posterior intestine but did not significantly change NKA activity in the anterior intestine which remained higher than that in the posterior intestine. Intestinal Cl- and water uptake, which was observed in ex vivo preparations of anterior and posterior intestine under both symmetric and asymmetric conditions, were higher in juveniles than in larvae and were similar in magnitude of those of teleost fish. Inhibition of NKA by ouabain in ex vivo preparations inhibited intestinal water absorption by 64 %. Our results suggest drinking and intestinal ion and water absorption are important to osmoregulation in SW, and that preparatory increases in intestinal NKA activity are important to the development of salinity tolerance that occurs during sea lamprey metamorphosis.