In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands of Squalus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170 mM KCl gradient was initially about five-fold higher than in the presence of a 170 mM KNO3 gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4 mM bumetanide and 40% by 0.8 mM N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170 mM NaCl gradient than in the presence of a 170 mM NaNO3 gradient. The effect of chloride was completely abolished by 0.4 mM bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound 3[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparent KD of 3.5 x 10(-6) M and a number of sites n of 104 pmol/mg protein. At 0.8 microM, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600 mM or when 600 1 mM bumetanide or furosemide was added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.