The ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by a variety of polyamines, including spermine, spermidine, and poly(arginine). The effects of spermine on the ATPase are highly specific. It has no effect on the affinity of the ATPase for Ca2+ or ATP, and no effect on the rate of phosphorylation by ATP. When the ATPase is phosphorylated with Pi in the presence of dimethyl sulfoxide at pH 6.0, and then dephosphorylation is induced by dilution in buffer at pH 7.5 in the absence of dimethyl sulfoxide, spermine is found to have no effect on the rate of dephosphorylation. If the ATPase is phosphorylated with [gamma-32P]ATP and the rate of loss of radiolabeled phosphoenzyme is measured following the addition of unlabeled ATP, spermine is found to decrease the rate of loss of radiolabel, consistent with an effect of spermine on the rate of the Ca2E1P-->E2P step. Direct measurement confirms that spermine decreases the rate of dissociation of Ca2+ from the phosphorylated ATPase (Ca2E1P-->E2P), with the decrease in the rate of this step explaining the inhibition of ATPase activity. Spermine also increases the equilibrium constant E1/E2 and inhibits phosphorylation of the ATPase by Pi by competition with the Mg2+ essential for the reaction. It is suggested that spermine could bind to the site on the Ca(2+)-ATPase that interacts with phospholamban.