Ca influx and removal mechanisms are vitally important to Ca homeostasis and thus the correct functioning of the myometrium. Both Na/Ca exchange and PMCA have been implicated in Ca extrusion following uterine stimulation. PMCA is encoded by four genes (PMCA 1-4), with PMCA 1 and 4 isoforms found in most tissues. The aim of this work was to investigate the functional effects of knocking out PMCA 4.Wild type, heterozygous, and homozygous knockout PMCA 4 black 6129 mice were killed humanely under CO2 anaesthesia, and the uterus removed. Small strips of myometrium were dissected and attached to a force transducer. After establishing contraction to high K depolarisation (120 mM), the tissues were then placed in either 0-Ca (1mM EGTA) control solution, or 0-Ca 0-Na (1mM EGTA) solution, to impair Na/Ca exchange, and the half time of force decline calculated. The tissues were then stimulated with carbachol (100 µM) for 20s, again in either 0-Ca or 0-Ca 0-Na solution, and the amount of force produced by SR Ca release compared. The experiments were performed and analysed blindly. Western blotting was used to test for possible compensatory upregulation of the Na/Ca exchanger. Mean ± Standard error are given, and significance was tested using the paired t-test. Control mouse myometrium produced regular, phasic contractions proceeded by intracellular Ca2+ transients (n=20), and carbachol and high K increased the Ca2+ signals and contractions. There were no significant differences in the rate of fall of force in any group in 0-Ca solution (n=15). However upon removal of extracellular Ca and Na, the rate of fall was significantly reduced in the homozygous KO mice (37.73±7.8% of KCL contraction) and the heterozygous mice (14.19±4.4%) compared to the wild type (79.45±3.7%). There was no statistical difference observed between the force produced by the release of SR Ca2+ in the three genotypes. There was no observed difference in the expression of the Na/Ca exchanger in the PMCA 4 knock out mice when compared to the wild type (n=2). These data suggest that both Na/Ca exchange and PMCA are responsible for lowering Ca and force in the myometrium following stimulation. If PMCA activity is impaired, as in the KO mice, then increased Na/Ca exchange activity can compensate, despite no observed increase in the expression of the protein. The unchanged response to carbachol in the KO mice suggests that the SR has taken up no more Ca than in control mice, and is therefore functionally unaffected by the changes in plasmalemmal Ca extrusion. If the system is challenged in the homozygous KO mice, e.g. by reducing external Na, then extrusion and relaxation of force are greatly impaired.