The sarco/endoplasmic reticulum calcium ATPases (SERCA) play a crucial role in regulating free cytosolic calcium (Ca²⁺) concentration in diverse cell types. It has been shown that recombinant SERCA3 exhibits low apparent affinity for Ca²⁺ when measured in heterologous systems; however, the Ca²⁺ affinity of native SERCA3 in an endogenous setting has never been examined. Such a measurement is complicated because SERCA3 is always co-expressed with the housekeeping isoform SERCA2b. We examined the properties of endogenous human SERCA3 and SERCA2b using a fluorescence-based assay for monitoring continuous Ca²⁺ uptake into microsomes. The kinetic parameters were derived using a cooperative two-component uptake model for Ca²⁺ activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody, PL/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca²⁺ affinity for SERCA3 compared to SERCA2b (1.10 ± 0.04 µM versus 0.26 ± 0.01 µM, respectively), and validated the two-component uptake model using mixtures of recombinant protein isoforms. Then, we determined apparent Ca²⁺ affinity for SERCA proteins present endogenously in cultured Jurkat T-lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca²⁺ affinity for SERCA3 in these two preparations was 1.04±0.07 µM and 1.1±0.2 µM, and for SERCA2b was 0.27±0.02 µM and 0.26±0.01 µM, respectively. Our data demonstrate, for the first time, that SERCA3 expressed in situ displays inherently lower affinity for Ca²⁺ in comparison with other SERCA isoforms. The physiological implications of this finding are discussed.