The slow/cardiac isoform of the sarcoplasmic reticulum Ca2+-ATPase plays an important role in cardiac muscle Ca2+-homeostasis. To determine the native configuration of the SERCA2 ion pump, a chemical cross-linking analysis of heart microsomes was performed. Using one- and two-dimensional immunoblotting following incubation with the hydrophilic probe bis-sulfosuccinimidyl suberate or the hydrophobic crosslinker dithiobis-succinimidyl-propionate, we demonstrate here that SERCA2 forms high-molecular-mass aggregates. In contrast to the Na+/Ca2+-exchanger, Ca2+-ATPase clusters can be stabilized by hydrophilic 1.2 nm crosslinkers and are sensitive to chemical reduction. Hence, the native form of this important Ca2+-regulatory membrane protein involved in cardiac muscle relaxation appears not to exist as a monomeric ion pump unit. Protein-protein interactions might play an important role in the physiological functioning of this Ca2+-ATPase isoform, as has previously been shown for skeletal muscle Ca2+-pumps, Ca2+-binding proteins and Ca2+-channels.