We have detected regulatory interactions between the sarcoplasmic reticulum calcium-transporting ATPase (Ca-ATPase, aka SERCA) and its inhibitory subunit sarcolipin (SLN) from skeletal muscle. SLN inhibits SERCA by uncoupling calcium transport, reducing catalytic efficiency from two to one calcium ion transported per ATP molecule hydrolyzed. Phosphorylation of SLN is the switch that relieves SERCA inhibition; it is unknown whether SLN phosphorylation disrupts the inhibitory complex (dissociation model) or causes a structural rearrangement (subunit model). Crosslinking assays indicate that calcium binding to SERCA disrupts interaction with SLN, suggesting that SLN dissociates once per catalytic cycle during the key step of calcium uncoupling (Toyoshima, Nature 2013). We previously detected oligomeric interactions between the two proteins using fluorescence resonance energy transfer (FRET) microscopy, finding that that SLN binds to SERCA in a 1:1 binary complex in the presence of micromolar calcium and millimolar ATP (Autry, JBC 2011). Here we used FRET spectroscopy to detect regulatory interactions in the presence or absence of calcium and/or ATP, finding that the two proteins show similar binding in calcium-bound and calcium-free states of SERCA, regardless of nucleotide occupancy. We also assayed SLN-SERCA interaction by column chromatography on anion-exchange and nucleotide-mimetic resins; results indicate that SLN remains bound to SERCA following detergent solubilization in the presence of calcium. We conclude that calcium does not dissociate SLN from SERCA, a result consistent with SLN-mediated uncoupling of calcium transport. Addition of calcium/calmodulin-dependent kinase did not disrupt SLN-SERCA interaction; thus, we propose that the non-dissociative subunit model is the molecular mechanism that mediates SLN regulation of SERCA. Acknowledgments: FRET was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Greg Gillispie, President). This work was funded by NIH grants to DDT (R01-GM27906, P30-AR0507220, T32-AR007612).