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Sodium uptake by rainbow trout gills has been investigated with a small-volume system enabling rapid, successive flux measurements in different solutions. Sodium influx obeys a Michaelis-Menten type relation, with a K(m) of 0.46 mM, and uptake proceeds unimpaired in the absence of penetrating counter-ions. This suggests a coupled cation exchange. Ammonia output is about the same as the Na(+) influx when external [Na(+)] is 1 mM, but at higher or lower Na(+) influxes, the correlation does not hold. A progressive downward shift in the pH of the irrigating medium as Na(+) influx increases indicates that the exchanging cation is hydrogen. In support of this, acetazolamide, which inhibits Na(+) uptake, also prevents the downward pH shift. The potential across the gill is about 10 mv, body fluids positive, in NaCl solutions up to 10 mM, and is little affected by changes in Na(+) concentration below that. Finally, evidence for locating the rate-limiting step at the outer membrane of the epithelium is presented.