Cardiac connexin 43 (Cx43) is involved in infarct propagation, and uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43-dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43-dephosphorylation. In addition to the sarcolemma, Cx43 is also present in cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1, PP2A and PP2B and the role of such interaction for Cx43-phosphorylation in preconditioned myocardium. Infarct size (% of the area at risk) in left ventricular (LV) anterior myocardium of Göttinger minipigs subjected to 90 min low-flow ischemia and 120 min reperfusion was 23.1±2.7 (n=7, non-preconditioned (NIP) group) and was reduced by IP to 10.0±3.2 (n=6, p<0.05). Mitochondrial and gap junctional Cx43-dephosphorylation increased after 85 min ischemia in NIP myocardium, whereas Cx43-phosphorylation was preserved with IP. PP2A and PP1, but not PP2B were detected by Western blot analysis in LV myocardium. Cx43 co-precipitated with PP2A, but not with PP1. Although total PP2A immunoreactivity (confocal laser scan) was increased to 154±24% and 194±13% of baseline (p<0.05) after 85 min ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between groups. The amount of PP2A co-immunoprecipitated with Cx43 remained unchanged. Only PP2A co-precipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2A is not involved in the prevention of the ischemia-induced Cx43-dephosphorylation by IP.