The effects of 4-hydroxy-3-nitrophenylglyoxal (HNPG), on the binding of eosin-5-maleimide (EMA), and diethyl pyrocarbonate (DEPC) to the anion transport system in the human red blood cell membrane, have been investigated. HNPG is a reversibly binding, arginine-specific, anion transport competitive inhibitor, known to act on the anion binding site. The EMA reaction site is an external facing lysine residue (Lys-430) in the 17 kDa transmembrane segment. The DEPC reaction site is an intracellular histidine (His-819) in the 35 kDa fragment. The results show that inhibition of the transport system with EMA increases in presence of HNPG to about 2.3 times. This finding suggests a positive cooperativity between the HNPG and EMA binding site and give evidence that the essential arginine is either nearby or allosterically linked to Lys-430. The inhibition of the cells with DEPC was nearly unchanged or slightly decreased in the presence of 10 mM HNPG. These results suggest that the intracellular His-residue which reacts with DEPC is not a part of the transport pathway. Our experiments with 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) have shown that its affinity to the transport system does not change after pre-treatment with phenylglyoxal (PG). We also found that the binding of [14C]phenylglyoxal (PG) to band 3 reduces significantly in presence of chloride. This is another evidence for the direct involvement of arginine residues in substrate binding.