The Sarco/Endoplasmic Reticulum (SR/ER) Ca²⁺-ATPases (SERCAs) transport cytosolic Ca²⁺ to the lumen of the SR/ER, the major intracellular Ca²⁺-store. Among all SERCA isoforms, the housekeeping isoform SERCA2b draws specific attention due to its extended carboxyl tail which comprises an eleventh transmembrane segment (TM11). This unique SERCA2b tail confers to the proteins' distinct functional properties when compared to the muscle-specific SERCA2a splice variant. SERCA2b displays a two-fold higher affinity for Ca²⁺ and a two fold lower catalytic turnover rate than SERCA2a. With exception of the 2b tail the two pumps share the same amino acid (aa) sequence. The 2b tail (49aa) consists of a cytosolic (20aa), a transmembrane (17aa) and a luminal (12aa) part. To explore the function of the 12aa-long luminal tail we mutated each of the 12aa in turn either to alanine or to an early stop. These 24 mutants were overexpressed in COS-cells and microsomes were prepared. Ca²⁺ -dependent ATPase measurements on the 12 different microsome preparations of the alanine scanning revealed that the last 4 aa (-MFWS) of the luminal tail are involved in increasing the Ca²⁺ affinity of the pump. These results suggest that the last 4 amino acids of the SERCA2b tail can function as a hook to interact with luminal domains more upstream of the pump. In addition, Ca²⁺ -dependent ATPase measurements on the 12 truncated SERCA2b mutants indicate that besides the luminal part, the TM11 segment is also an important determinant of the enzymatic properties of the pump.