Active transcellular transport of NaCl in thick ascending limb of Henle's loop (TAL) consists of primary active transport of Na+ and secondary active transport of Cl-. The Na,K-pump maintains a low electrochemical potential for Na+ in the cytoplasm. The Na,K,Cl-cotransport system couples the entry of Cl- across the lumen membrane to entry of Na+ along its gradient. In mammalian kidney the TAL is concentrated in the red outer medulla and this tissue has exceedingly high concentrations of Na,K-pump sites, comparable with those in salt glands of birds and fishes. With the red outer medulla as starting material we developed procedures for purification of the Na,K-pump in membrane-bound form. The pump protein can be immobilized in two-dimensional membrane crystals by stabilization of the E2 form with vanadate or phosphate. The minimum functional protein unit of both membrane-bound and soluble Na,K-ATPase is an alpha beta-dimer (alpha-subunit Mr 84,000-106,000 and beta-subunit Mr 32,000-38,000 plus carbohydrate). Binding data, proteolytic digestion and fluorescence analysis show that both Na,K-induced and ATP-dependent conformational transitions of the alpha-subunit can be demonstrated in preparations consisting of soluble and fully active alpha beta-units. This is consistent with the notion that a single alpha beta-unit can catalyse the whole series of intermediary reactions and cation transport in the membrane. The Na,K,Cl-cotransport system can be identified by assaying bumethanide-sensitive ion fluxes in membrane vesicles from the outer renal medulla. Using equilibrium binding with [3H]bumethanide as an assay the cotransporter has been partially purified to capacities of 600-1,000 pmol/mg of protein. A polypeptide of Mr 34,000 is covalently labelled with [3H]bumethanide by direct photolysis. The [3H]bumethanide binding protein is not extracted by nonionic detergent except at high ionic strength, suggesting that the Na,K,Cl-cotransport protein is associated with cytoskeleton components. This association may be important for control of the entry of NaCl into the cytoplasm and for cellular regulation of the rate of active transport of NaCl across the tubule cells in the TAL.