The effects of a new potent and selective inhibitor the Na⁺/Ca²⁺ exchange, SEA0400 (SEA), were studied in internally dialyzed squid giant axons, both from the extra and intracellular side, on steady-state outward (forward exchange), inward (reverse exchange) and Ca²⁺/Ca²⁺ transport exchange modes. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: i) it increases the synergic (Na⁺_i + H⁺_i) inactivation; ii) is antagonized by ATP and intracellular alkalinization; and, iii) is enhanced by intracellular acidification even in the absence of Na⁺. Inhibition by SEA is still present even after one hour of its removal from the experimental solutions, whereas removal of the co-interacting agent of inhibition, Na⁺_i and H⁺ _i, even in the continuous presence of SEA, releases inhibition thus indicating that SEA facilitates the reversible attachment of the natural H⁺_i and Na⁺ _i synergic inhibitors. Based of a recent model for the squid Na⁺/Ca²⁺ exchange regulation (DiPolo and Beauge, J. Physiol., 593: 791-893, 2002), we suggest that SEA acts on the (H⁺ _i + Na⁺_i)-inactivation process, and can interact with the Na⁺-free and Na⁺-bound protonized carrier. Protection by ATP concurs with the antagonism of the nucleotide with the (H⁺_i + Na⁺_i) synergic inhibition.