Sarcoplasmic reticulum ATPase has been found to cleave the ATP analog adenyl-5'-yl imidodiphosphate in a calcium-dependent reaction. The reaction products were determined by 31P NMR to be inorganic phosphate and adenyl-5'-yl phosphoramidate (AMP-PN). AMP-PNP hydrolysis, like ATP hydrolysis, drives active Ca2+ accumulation by sarcoplasmic reticulum vesicles.