Evidence for intraluminal Ca++ regulatory site defect in sarcoplasmic reticulum from malignant hyperthermia pig muscle. Malignant hyperthermia (MH) is a pharmacogenetic disease of humans and various animal species that predisposes to a life-threatening, anesthetic agent-induced syndrome. MH is thought to be a consequence of abnormal, sustained increases in myoplasmic Ca++ and sarcoplasmic reticulum (SR) membranes from MH muscle have been shown to have a Ca++ release channel defect. In the present study we have tested a hypothesis that the abnormal Ca++ release mechanism in MH can be expressed when Ca++ is loaded in the presence of pyrophosphate. SR membrane vesicles isolated from normal and MH pig muscle were loaded with Ca++ in the presence and absence of pyrophosphate until Ca(++)-induced Ca++ release occurred. Under both circumstances the threshold amount of Ca++ loaded until Ca++ release occurred was lower in the SR from MH pig skeletal muscle. This difference in amount of Ca++ preload is not explained by results obtained comparing rates of Ca++ uptake, number of ryanodine binding sites or the amounts of calsequestrin among SR vesicles from MH and normal muscle. We conclude from this study that use of pyrophosphate for Ca++ loading does not ablate the abnormal Ca++ release in SR from MH muscle, suggesting the study can be done on small amounts of SR from biopsied human muscle. The data also suggest that abnormality in an intraluminal, low affinity Ca++ binding site regulating Ca++ release occurs in the SR membrane of MH pig muscle. 