We have measured the microsecond rotational motions of the sarcoplasmic reticulum (SR) Ca-ATPase as a function of enzyme-specific ligands, including those that induce active calcium transport. We labeled the Ca-ATPase with a maleimide spin probe and detected rotational dynamics using saturation-transfer electron paramagnetic resonance (ST-EPR). This probe’s ST-EPR spectra have been shown to be sensitive to microsecond protein rotational motion, corresponding to large-scale protein rotations that should be affected by changes in the enzyme’s shape, flexibility, protein-protein interactions (oligomeric state), and protein-lipid interactions. We found that the motions of the enzyme-nucleotide and the enzyme-nucleotide/Ca states are indistinguishable from the motions in the absence of ligands. Rotational mobility does decrease in response to the addition of DMSO, a solvent that inhibits Ca-ATPase activity and stabilizes the phosphoenzyme. However, the addition of phosphate to form phosphoenzyme, in the presence or absence of DMSO, does not change the motions significantly. During the steady state of active calcium transport, the microsecond rotational mobility is indistinguishable from that of the resting enzyme. In order to detect any transient changes in mobility that might not be detectable in the steady state and to improve the precision of steady-state measurements, we photolyzed caged ATP with a laser pulse in the presence of calcium and detected the ST-EPR response from the spin-labeled enzyme, with a time resolution of 1 s. No significant change in the ST-EPR signal was observed, indicating that the effective rotational correlation time does not change by more than 10% in the transient or steady-state phases of the Ca-ATPase cycle. While protein motion has been shown to be important to the function of the Ca-ATPase, this study indicates that changes in the microsecond protein rotational mobility, which would be caused by changes in the enzyme’s shape, flexibility, oligomeric state, or protein-lipid interactions, do not occur as part of the calcium transport cycle. This does not rule out (1) transient-phase effects lasting less than 1 s, (2) short-lived effects that occur after the rate-limiting transition, or (3) rearrangements within an oligomeric unit that do not affect the overall rotational mobility.