Others have shown that H₂DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO₃ exchanger AE1. Here we mutated K558, K559 and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (-160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent K_i for reversible DIDS binding increased in the sequence RRMIR<KKMIK(wt, ~40 µM)<NKMIKNKMINKKMIN<KNMINKNMIK<NNMIK<NNMIN(~400 µM)<DDMID<EEMIE(~800 µM). Thus, the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80-91% for the first five constructs, and 66% for NNMIN. Thus, DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.