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Adipose tissue-derived mesenchymal stem cells (ASCs) are a promising stem cell source for cell transplantation. We demonstrate that undifferentiated ASCs display robust oscillations of intracellular calcium [Ca2+]i which may be associated with stem cell maintenance since oscillations were absent in endothelial cell differentiation medium supplemented with FGF-2. [Ca2+]i oscillations were dependent on extracellular Ca2+ and Ca2+ release from intracellular stores since they were abolished in Ca2+-free medium and in the presence of the store-depleting agent thapsigargin. They were inhibited by the phospholipase C antagonist U73,122, the inositol 1,4,5-trisphosphate (InsP3) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) as well as by the gap-junction uncouplers 1-heptanol and carbenoxolone, indicating regulation by the InsP3 pathway and dependence on gap-junctional coupling. Cells endogenously generated nitric oxide (NO), expressed NO synthase 1 (NOS 1) and connexin 43 (Cx 43). The nitric oxide NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine methyl ester (L-NAME), 2-ethyl-2-thiopseudourea, and diphenylene iodonium as well as si-RNA-mediated down-regulation of NOS 1 synchronized [Ca2+]i oscillations between individual cells, whereas the NO-donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) as well as the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) were without effects. The synchronization of [Ca2+]i oscillations was due to an improvement of intracellular coupling since fluorescence recovery after photobleaching (FRAP) revealed increased reflow of fluorescent calcein into the bleached area in the presence of the NOS inhibitors DPI and L-NAME. In summary our data demonstrate that intracellular NO levels regulate synchronization of [Ca2+]i oscillations in undifferentiated ASCs by controlling gap-junctional coupling. J. Cell. Physiol. 226: 1642–1650, 2011. © 2010 Wiley-Liss, Inc.