Experiments were performed to determine whether the organic Ca²⁺ channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) Ca²⁺ uptake by directly inhibiting the light SR Ca²⁺-ATPase. We have previously shown that at 10 µM, D-600 inhibits LSR ATP-dependent Ca²⁺ uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR Ca²⁺-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas Ca²⁺ 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 Ca²⁺-ATPase demonstrated that D-600 decreased the thermal stability of Ca²⁺ transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 µM did not have any leaking effect of Ca²⁺ on the Ca²⁺-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca²⁺-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the Ca²⁺ blocker D-600 inhibits the SR Ca²⁺ pump specifically by interacting with the transmembrane Ca²⁺-binding domain of the Ca²⁺-ATPase.