In seawater-acclimated rainbow trout (Oncorhynchus mykiss), base secretion into the intestine is a key component of the intestinal water absorption that offsets osmotic water loss to the marine environment. Acid-base balance is maintained by the matched excretion of acid equivalents via other routes, presumably the gill and/or kidney. The goal of the present study was to examine acid-base balance in rainbow trout upon transfer to more dilute environments, conditions under which base excretion into the intestine is predicted to fall, requiring compensatory adjustments of acid excretion at the gill and/or kidney if acid-base balance is to be maintained. Net acid excretion via the gill/kidney and rectal fluid, and blood acid-base status were monitored in seawater-acclimated rainbow trout maintained in seawater or transferred to iso-osmotic conditions. As predicted, transfer to iso-osmotic conditions significantly reduced base excretion into the rectal fluid (by ~48%). Transfer to iso-osmotic conditions also significantly reduced the excretion of titratable acidity via extra-intestinal routes from 183.4 ± 71.3 to -217.5 ± 42.7 μmol kg(-1) h(-1) (N = 7). At the same time, however, ammonia excretion increased significantly during iso-osmotic transfer (by ~72%) so that the apparent overall reduction in net acid excretion (from 419.7 ± 92.9 to 189.2 ± 76.5 μmol kg(-1 )h(-1); N = 7) was not significant. Trout maintained blood acid-base status during iso-osmotic transfer, although arterial pH was significantly higher in transferred fish than in those maintained in seawater. To explore the mechanisms underlying these adjustments of acid-base regulation, the relative mRNA expression and where possible, activity of a suite of proteins involved in acid-base balance were examined in intestine, gill and kidney. At the kidney, reduced mRNA expression of carbonic anhydrase (CA; cytosolic and membrane-associated CA IV), V-type H(+)-ATPase, and Na(+)/HCO(3) (-) co-transporter were consistent with a reduced role in net acid excretion following iso-osmotic transfer. Changes in relative mRNA expression and/or activity at the intestine and gill were consistent with the roles of these organs in osmotic rather than acid-base regulation. Overall, the data emphasize the coordination of acid-base, osmoregulatory and ionoregulatory processes that occur with salinity transfer in a euryhaline fish.