We have studied the effect of the endogenous inhibitor of the Na+ and Ca2+ pumps, HHIF, on sarcoplasmic reticulum (SR) vesicles. The effect of HHIF on the SR Ca2+,Mg(2+)-ATPase activity shows a biphasic pattern. Low HHIF concentrations activate the Ca2+,Mg(2+)-ATPase by dissipation of Ca2+ gradient across the SR membrane. Higher concentrations irreversibly inhibit this activity following a slow kinetic process both in intact SR membranes and in purified Ca2+,Mg(2+)-ATPase. Differential scanning calorimetry shows that the Ca2+,Mg(2+)-ATPase is denatured after incubation with HHIF concentrations which produced full inhibition of its activity. Micromolar Ca2+ and millimolar Mg2+ ADP protect against the irreversible inhibition of the Ca2+,Mg(2+)-ATPase by HHIF. The concentration of HHIF which produces 50% inhibition depends upon SR membrane concentration and upon the lipid:protein ratio in purified Ca2+,Mg(2+)-ATPase. From this we have obtained a partition coefficient for binding of HHIF to SR membranes of 0.6 (microgram SR protein/ml)-1.