SERCA1a and SERCA2a have been used interchangeably in mutagenic and structural analyses of the PLB-SERCA interaction, since in vitro studies have shown their functional inhibition by PLB is equivalent. To quantify the quaternary structure and binding energetics of PLB binding to SERCA isoforms, fluorescence resonance energy transfer (FRET) from Cer-SERCA1a or Cer-SERCA2a to YFP-PLB was measured in live AAV-293 cells. FRET efficiency increased with increasing protein expression level to a maximum of 28.8% for PLB-SERCA1a and 28.1% for PLB-SERCA2a, suggesting the complexes have the same quaternary conformation. Unexpectedly, the data also revealed that PLB has a 2.6 fold higher apparent affinity for SERCA1a relative to SERCA2a. To test whether the observed difference in affinity arises from differential distributions of SERCA E1/E2 enzymatic substates, cells were treated with 1mM EGTA and 0.5uL/mL calcium ionophore A23187. Under these conditions, PLB still showed greater affinity for SERCA1a over SERCA2a, suggesting that the differential affinities are intrinsic properties of the SERCA isoforms. The data suggest that PLB preferentially binds SERCA1a over SERCA2a, which may be an important strategic consideration for therapeutic overexpression of SERCA isoforms in cardiac muscle.