The Na+-Ca2+ exchanger plays an important role in cardiac contractility by moving Ca2+ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na+-Ca2+ exchange activity were determined. Exchange activity was assessed as 45Ca2+ uptake into Na+-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function.