The inactivation of the renal outer cortical brush-border membrane D-glucose transporter by the covalent carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) is studied by monitoring its effects on sodium-dependent phlorizin binding to the active site of the carrier. In the presence of EEDQ, this component of phlorizin binding decreases exponentially and irreversibly with time. The order of this inactivation reaction is very close to 1, indicating that EEDQ modifies the transporter at a single essential site. This site can be partially protected by glucose and by other substrates of the transporter and completely protected by phlorizin, a nontransported competitive inhibitor. By contrast, sodium, a co-transported activator, has no protective effect. The concentration dependence of the protection provided by glucose and phlorizin indicates that the site of action of EEDQ is at or closely related to the substrate binding site on the carrier. The effects of EEDQ on the transporter are mimicked by another carboxyl specific reagent, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate. The rate of inactivation of the transporter by EEDQ increases dramatically with decreasing pH, consistent with the hypothesis that the rate-limiting step in the inactivation process is a reaction with an essential carboxyl group. The properties of this group indicate, however, that it is distinct from the carboxyl group proposed by others as forming (a part of) the sodium binding site of sodium-coupled sugar carriers.