3,5-Di-t-butyl-4-hydroxyanisole (DTBHA) increased in a concentration-dependent manner (calculated pEC(50) = 4.55 +/- 0.18 M) the oxalate-stimulated Ca(2+)-pumping rate of rat skeletal muscle sarcoplasmic reticulum (SR) vesicles. Kinetic analysis of this effect suggested that the activation of SR Ca(2+)-ATPase operated by (DTBHA) was of both mixed and non-competitive type with respect to ATP in the range of concentrations 0.1-0.5 mM and above 1 mM, respectively; furthermore, it was independent of the free Ca(2+) concentrations. This indicated that the enzyme activation took place through the acceleration of the enzyme-substrate complex breakdown. Moreover, it appeared that its target site was cyclopiazonic acid sensitive. The uncommon ability of (DTBHA) to upregulate SR Ca(2+) uptake is of interest in view of its possible use for treating pathological conditions characterised by cell Ca(2+) overload as well as genetic disorders where SR Ca(2+) homeostasis is altered.