Na+,K+-ATPase is responsible for maintaining the cross-membrane Na+ and K+ gradients of animal cells. This P-type ATPase works via a complex transport cycle, during which it binds and occludes three intracellular Na+ ions and then two extracellular K+ ions, which it then releases on the other side of the membrane. The cation pathway through the protein, and the structures responsible for occluding cations inside the protein, have not yet been definitely identified. We used cysteine mutagenesis to explore the accessibility and the role of five conserved residues in the short third extracellular loop, between the fifth and the sixth transmembrane helices. The P801C and L802C mutants were not affected by extracellular sulfhydryl reagents. The presence of cysteine residues at three positions (G803C, T804C and V805C) conferred sensitivity to omeprazole, a known inhibitor of the gastric proton pump, and to [2-(trimethylammonium)-ethyl]methanethiosulphonate bromide (MTSET). The effects of omeprazole and MTSET were modulated by the presence of extracellular K+, indicating that the accessibility of these positions depended on the conformational state of the protein. MTSET binding to cysteine at position 803 partially inhibited the Na+,K+-pump function by decreasing its affinity towards extracellular K+, suggesting a restriction of the access of extracellular K+ ions to their binding sites. In contrast, MTSET binding to cysteine at position 805 partially inhibited the Na+,K+-pump function by reducing its maximum turnover rate, probably by slowing a rate-limiting conformational change. These residues occupy positions that are critical for either the cation pathway or the conformational modifications.