Experiments were performed to determine whether the organic Ca(2+) channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) Ca(2+) uptake by directly inhibiting the light SR Ca(2+)-ATPase. We have previously shown that at 10 microM, D-600 inhibits LSR ATP-dependent Ca(2+) uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR Ca(2+)-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas Ca(2+) binding and translocation are associated with the transmembrane domain (18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermal inactivation experiments using the SR Ca(2+)-ATPase demonstrated that D-600 decreased the thermal stability of Ca(2+) transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 microM did not have any leaking effect of Ca(2+) on the Ca(2+)-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca(2+)-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the Ca(2+) blocker D-600 inhibits the SR Ca(2+) pump specifically by interacting with the transmembrane Ca(2+)-binding domain of the Ca(2+)-ATPase.