We have recently reported that ATP up-regulation of the Na+/Ca2+ exchanger in the invertebrate squid nerve preparation take place as a result of drastic alterations in the Na+i-H+i-Ca2+i interactions with the large intracellular cytoplasmic loop of the exchanger (DiPolo & Beaugé, 2002). In this work we explored the mechanisms involved in the second main metabolic pathway of exchange regulation induce by phosphoarginine (PA). For this study we have used: a) intracellular dialyzed squid axons to measure Na+o-dependent 45Ca2+ efflux (Na+o-Ca2+i exchange), Ca2+o-dependent 45Ca2+ efflux (Ca2+o-Ca2+i exchange), Ca2+i-dependent 22Na+ efflux (Na+o -Na+i exchange) and Ca2+o-dependent 22Na efflux (Ca2+o-Na+i exchange) and b) squid optic nerve membrane vesicles to measure the Na+-gradient dependent 45Ca2+ uptake. Our results show the following characteristics of the PA regulation: 1) unlike ATP, it is not related to Na+i-H+i-Ca2+i interactions with intracellular "regulatory" loop; 2) it takes place away from that loop since PA effect remains unaltered after loop removal with chymotrypsine; and 3) modifies the affinity of the intracellular transport sites with a preferential increase in the affinity for Cai over that for Nai. We expanded our previous model of metabolic regulation of the Na+/Ca2+ exchanger by adding to the original "ATP region" a new zone, the "PA region" which is topologically related to the intracellular transporting sites for Na+i and Ca2+i. The favored increase in the forward (Na+o-Ca2+i exchange) reaction induce by PA over the other partial reactions, makes this metabolic pathway ideal for causing large increments in Ca2+ extrusion from the cell.