Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store-operated Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release-activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+ -dependent inactivation, inhibition/potentiation by 2-aminoethyl diphenylborinate and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2- and Orai3-mediated CRAC current (ICRAC ) by intracellular pH (pHi ). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi . Orai1-mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+ -dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi , whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1-Orai3 chimeras suggests that pHi dependence of Orai1 resides in both the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1. KEY POINTS: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)-mediated Ca2+ release-activated Ca2+ current (ICRAC ) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation. The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pHi ). The amplitude of Orai2 current is affected by the changes in pHi  similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca2+ -dependent inactivation of Orai2 is unaffected by acidic pHi . In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pHi  changes. Domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pHi . Reduction of ICRAC dependence on pHi seen in a STIM1-independent Orai1 mutant suggested that some parts of STIM1 are also involved in ICRAC modulation by pHi .