Ca²⁺ plays an essential role in the hearing process. It enters stereocilia of hair cells through mechanoelectrical transduction (MET) channels opened by the deflection of the hair bundle, and is exported back to endolymph that bathes the apical portion of apical cells by an unusual splicing isoform (w/a) of the PMCA2 pump. The w/a isoform carries inserts at sites A and C. The C insert induces premature truncation of the pump. The w/a isoform of PMCA2 is the only means available to stereocilia to export Ca²⁺. Ablation or missense mutations of the pump cause deafness, as described for the first time in G283S mutation of the deafwaddler (dfw) mouse. A novel deafness-inducing missense mutation of PMCA2 (G293S), has been identified in a human family. The wild type PMCA2 w/a isoform and its G283S and G293S mutants were overexpressed in CHO cells. The other splice variants of PMCA2 (w/b, z/a, z/b) were also expressed as control. Recombinant aequorin was used to monitor Ca²⁺. At variance with the other PMCA2 isoforms the w/a variant became activated only marginally when exposed to a Ca²⁺ pulse induced in CHO cells by InsP3. The G293S and G283S mutations did not compromised firther the already poor ability of the w/a variant to became rapidly activated by Ca²⁺ pulse, but delayed the longer term dissipation of the Ca²⁺ transients. In organotypic cultures, Ca²⁺ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca²⁺ transients induced by Ca²⁺ uncaging was compromised in the dfw and also in the PMCA2 KO mice. A digenic mechanism was operational in the case of the human family with impaired hearing described here. The family was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation.