The STIM-Orai activating region (SOAR) of STIM1 is the minimal sequence necessary for Orai1 channel activation. Our previous work revealed that replacement of Phe-394 in SOAR with the dimensionally similar but polar histidine head group prevents Orai1 binding and gating. In the current study, we constructed YFP-SOAR-SOAR concatemers with a 21 amino acid linker between the SOAR units which allowed folding into functional dimers. We introduced the F394H mutation (FH) into either the first (N-terminal) or second (C-terminal) SOAR unit within the dimeric concatemer constructs. We found that each of these single-mutated concatemers was able to both bind to Orai1 and gate the Orai1 channel normally, the same as the wild-type concatemer. In contrast, a concatemer in which both SOAR units contained the F394H mutation (YFP-SOARFH-SOARFH), was devoid of Orai1 binding and gating activity. To ensure that the single mutated concatemers were not forming inter-concatemer dimers, we equally co-expressed the CFP-SOARWT-SOARWT and YFP-SOARFH-SOARFH concatemeric constructs in the same cell. While the CFP-SOARWT-SOARWT bound to and activated Orai1, it did not pull the YFP-SOARFH-SOARFH with it. Thus, the latter remained in the cytosol in these cells. This result is in contrast to co-expression of the monomeric CFP-SOARWT with YFP-SOARFH constructs. In this case, while the YFP-SOARFH expressed alone does not bind to or activate Orai1, the CFP-SOARWT clearly dimerizes with YFP-SOARFHmutant and pulls it to the PM. This also proves that he F394H mutation does not prevent SOAR-SOAR dimer formation. Overall, the results reveal a surprising new facet of the STIM1-Orai1 coupling interaction. Thus, although the SOAR dimer is likely the functional Orai1-actiavting unit, only one of the SOAR units within the SOAR dimer needs to be able bind to and activate the Orai1 channel.