Regulation of Ca2+/H+ antiporters may be an important function in determining the duration and amplitude of cytosolic Ca2+ oscillations. Previously the Arabidopsis Ca2+/H+ transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca2+ transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca2+/H+ antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca2+/H+ transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca2+ transport by other Ca2+/H+ antiporters. Ca2+/H+ antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca2+/H+ antiporter from any orgnism.