Accumulation of metabolites during myocardial ischaemia and their subsequent removal during reperfusion generates osmotic imbalances that can cause heart cells to swell. Volume regulatory mechanisms operate to minimise cell swelling during osmotic challenge, but uncontrolled swelling can lead to cell rupture and myocardial tissue damage. Phosphoinositide-3-kinases (PI3Ks), which phosphorylate a wide range of downstream targets, are activated during pre- and post-conditioning protocols that limit ischaemic tissue damage. Activation of PI3K is thought to be a primary mediator of such cardioprotective mechanisms [1]. This study tests the hypothesis that PI3K signalling in the heart is a key component of the volume recovery response following osmotic swelling. Limiting cell swelling during ischemia/reperfusion could contribute to the functional benefits of pre- and post-conditioning. Cardiac myocytes were isolated enzymatically from embryonic chick hearts (day 11) and cultured overnight. Single spherical myocytes were superfused with isosmotic Hepes-buffered saline solution (300mOsmol/L), then swollen by exposure to hyposmotic solution (180mOsmol/L, by reduction of [NaCl]). Digital images of the cells were captured at 1 minute intervals throughout the experiment, and myocyte volume was calculated from these two-dimensional images. Myocytes exposed to hyposmotic challenge demonstrated a classic regulatory volume decrease (RVD), swelling to 149+12% of control volume (mean+SD; n=15), then recovering to 128+15% of control (mean+SD) after 20mins in hyposmotic solution. This volume recovery failed in the presence of the PI3K inhibitor wortmannin (5nM); cells remained swollen throughout hyposmotic challenge at 147+9% of control volume (mean+SD; n=6) after 20mins. Lower concentrations of wortmannin did not prevent RVD (1nM; n=7). The structurally-distinct PI3K inhibitor LY294002 (1μM) also blocked RVD; cell volume was 146+5% of control volume (mean+SD; n=4) after 20mins in hyposmotic solution. Cell volume during sustained hyposmotic challenge was significantly bigger when PI3Ks were inhibited compared to controls (p<0.001; unpaired t-tests). The isoform-specific PI3Kγ inhibitor AS604850 (10nM) also prevented volume recovery; cell volume remained at 136% of control (n=2) throughout hyposmotic challenge. These data demonstrate that PI3K activity is an essential element in the regulatory response to cardiac cell swelling. This enzyme could be an upstream activator of MAP kinase signalling pathways or cytoskeletal rearrangements, both of which have been shown previously to be instrumental in RVD [2, 3]. This study provides evidence that the cardioprotective role of PI3K during post-conditioning may be mediated, in part, by preventing ischaemia/reperfusion-induced cell swelling and thereby limiting cell death in the myocardium.