The choline transport system of erythrocytes is reversibly inhibited by ethanol, n-butanol, n-hexanol, n-octanol, and n-decanol, but not by n-dodecanol. Each methylene group in the alkyl chain contributes 560 cal/mol to the free energy of binding at the inhibitory site. Inhibition results from the cooperative binding of two molecules of an alcohol, judging by the Hill coefficient n of 1.7-1.9. The mechanism of inhibition is noncompetitive, and the partition of the carrier between inward-facing and outward-facing forms is unaffected by the alcohols; it follows that the four main carrier forms, the inner and outer free carrier, and the inner and outer carrier-substrate complex, are equally susceptible to inhibition. Hexanol and decanol accelerate the reaction of N-ethylmaleimide with a thiol group in the inner carrier channel, but ethanol and butanol, at concentrations that inhibit transport by 70%, do not. The disproportionate effects on substrate transport and the N-ethylmaleimide reaction are most simply explained as the direct result of binding of alcohol molecules in different regions of the carrier, rather than as the indirect result of a disturbance in the structure of the lipid bilayer induced by the alcohols.