Two proteins were purified earlier from solubilized membranes of Ehrlich ascites cells by using a bumetanide-Sepharose affinity column. These proteins were proposed to be constituents of the Na-K-C1 cotransporter. However, the specificity of binding of bumetanide to the cotransporter was insufficient evidence for this proposal. We now have direct evidence that the purified protein contains components of the cotransporter. Antiserum raised against the bumetanide-binding proteins strongly inhibits Na-K-C1 cotransport measured by two independent methods. Cotransport was induced by hypertonic challenge and was measured as the bumetanide-sensitive portion of unidirectional C1 influx and as regulatory cell volume increase. In both assays, cotransport was strongly inhibited by the antiserum. Fab fragments of the antibodies inhibited cotransport to a similar extent.