Caloxin 2A1 is a novel inhibitor of the plasma membrane (PM) Ca 2+-pump [Am. J. Physiol. Cell Physiol. 280 (2001) C1027]. The PM Ca 2+-pump is a Ca 2+–Mg 2+-ATPase that expels Ca 2+ from cells to help them maintain low concentrations of cytosolic Ca 2+. Caloxin 2A1 inhibits Ca 2+–Mg 2+-ATPase in human erythrocyte leaky ghosts. Here we report that this inhibition is non-competitive with respect to the substrates Ca 2+ and ATP and the activator calmodulin. This was anticipated since the high affinity binding site for Ca 2+ and sites for ATP and calmodulin are intracellular whereas caloxin 2A1 is a peptide selected for binding to the second extracellular domain of the pump. Caloxin 2A1 also inhibited the Ca 2+-dependent formation of the acid stable 140 kDa acylphosphate intermediate from 32 P -γ-ATP. However, it did not inhibit the formation of the acylphosphate intermediate in the reverse direction—from 32 P -orthophosphate. Consistent with results on mutagenesis of transmembrane residues in the pump protein, we suggest that caloxin 2A1 inhibits conformational changes required during the reaction cycle of the pump.