Plasma membrane calcium ATPases (PMCAs) use energy derived from ATP to drive Ca²⁺ from the cytosol into the extracellular fluid. PMCAs act as a highly efficient primary route for removing intracellular Ca²⁺ ([Ca²⁺]_i) at sub-micromolar concentrations (Thayer et al. 2002). As the product of 4 genes (PMCA1-4) PMCAs are alternatively spliced to produce 20 identified splice variants. It is believed that alternative splicing may localise specific PMCA splice variants to synapses and that these splice variants may regulate synaptic transmission by controlling [Ca²⁺]_i at these sites (Strehler & Zacharias, 2001). Here we investigated the synaptic localisation of the neurone-specific PMCA2 splice variant PMCA2a and examined its function in the regulation of excitatory synaptic transmission in the CA3 region of organotypic hippocampal slices. Organotypic hippocampal slices were prepared from 7 day old Wistar rat pups (Stoppini et al. 1991) and were maintained for 10-14 days in vitro before they were used for electrophysiology or immunohistochemistry. Confocal fluorescence microscopy was carried out as previously described (Buckby et al. 2006) and co-localisation was quantified using Bio-Rad Lasersharp software. Whole-cell patch clamp recordings of evoked excitatory post-synaptic currents (EPSCs) from CA3 pyramidal neurones held at -70mV were carried out in the presence of 10µM bicuculline, 5µM CGP52432 and 0.5µM NBQX using an Axoclamp 2B patch clamp amplifier in voltage clamp mode. Using PMCA splice-variant specific antibodies we show that the PMCA2 splice variant PMCA2a is enriched in forebrain synaptosomes and co-localises with the pre-synaptic marker synaptophysin but not the post-synaptic marker PSD-95. Furthermore, PMCA2a co-localised at pre-synaptic terminals immunopositive for the excitatory pre-synaptic terminal marker VGLUT1 but not the inhibitory pre-synaptic terminal marker GAD-65 indicating that PMCA2a is localised specifically to excitatory pre-synaptic terminals. Whole cell patch clamp recordings showed that treatment of slices with the specific PMCA inhibitor carboxyeosin (CE) significantly enhanced paired pulse facilitation (PPF) lengthening the time course of PPF. For example CE treatment enhanced PPF with a 50ms inter-stimulus interval from a paired pulse ratio (EPSC2/EPSC1) of 2.13±0.19 (mean ± SEM) to 2.98±0.16 in 10μM CE-treated slices (n=6, p<0.01 paired t test) without affecting the mean amplitude of the 1st EPSC (p=0.78, n=6 paired t-test) indicating a pre-synaptic effect of PMCA inhibition. These results show that PMCA2a splice variants localise to excitatory pre-synaptic terminals and regulate excitatory synaptic transmission presumably by their role in controlling residual [Ca²⁺]_i.