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Band 3 is an integral membrane protein that ex-changes anions across the red cell membrane. Due to the abundance and the high turnover rate of the band 3 transport unit, the band 3 system is the most heavily used ion-transport system in a typical vertebrate or-ganism. Here we show that 36Cl NMR enables direct and specific observation of substrate C1- binding to band 3 transport sites, which are identified by a vari-ety of criteria: (a) the sites are inhibited by 4,4’-dinitrostilbene-Z,2’-dlsulfonate, which is known to in-hibit competitively Cl- binding to band 3 transport sites; ( b) the sites have affinities for 4,4‘-dinitrostil-bene-2,2’-disulfonate and C1- that are quantitatively similar to the known affinities of band 3 transport sites for these anions; and (c) the sites have relative affini