Physiological functions of the intracellular regulatory domains of the Na⁺/Ca²⁺ exchanger NCX1 were studied by examining Ca²⁺ handling in CCL39 cells expressing a low-affinity Ca²⁺ regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na⁺ inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (246-672). We found that D447V/D498I was unable to efficiently extrude Ca²⁺ from the cytoplasm, particularly during a small rise in intracellular Ca²⁺ concentration induced by the physiological agonist -thrombin or thapsigargin. The same mutant took up Ca²⁺ much less efficiently than the wild-type NCX1 in Na⁺-free medium when transfectants were not loaded with Na⁺, although it appeared to take up Ca²⁺ normally in transfectants preloaded with Na⁺. XIP-4YW and, to a lesser extent, 246-672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na⁺-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca²⁺ handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.