Phospholamban (PLN) is a 52-amino acid, integral membrane protein that interacts with and reversibly inhibits the activity of the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a). We have used site-directed mutagenesis to analyze the sites of interaction between PLN and SERCA2a. First, we used chimera formation between SERCA2a and SERCA3 (which is weakly inhibited by PLN) to determine the interacting residues in cytoplasmic sequences of SERCA2 and PLN. Then, we expressed SERCA2a with the transmembrane sequence of PLN and demonstrated that the sites of inhibitory interaction are located in transmembrane sequences of the two proteins. We proposed that a four-base circuit involving noninhibitory cytoplasmic and inhibitory transmembrane sites in PLN and SERCA2a best describes the interaction. Recently, we have used alanine-scanning mutagenesis to show an asymmetric distribution of function in the transmembrane domain of PLN--one helical face interacts with PLN molecules in a pentamer, and the other interacts with SERCA2a. Gain of function by mutation of PLN-interacting residues indicates that the inhibitory species of PLN is a monomer. Thus regulatory steps include PLN dissociation, PLN/SERCA2a inhibitory association, and PLN/SERCA2a dissociation induced by phosphorylation of PLN (in the noninhibitory cytoplasmic domain) or by binding of Ca2+ by SERCA2a (in the inhibitory transmembrane domain).