1 Nitric oxide (NO) is a key modulator of cellular Ca2+ signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca2+ entry (CCE) into HEK293 cells by impairment of mitochondrial Ca2+ handling. 2 Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 muM, respectively. 3 NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 muM), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 muM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 muM) failed to antagonize the inhibitory action of NO on CCE. 4 DEANO (1-10 muM) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca2+ levels, respectively, indicated that DEANO (10 muM) depolarized mitochondria and suppressed mitochondrial Ca2+ sequestration. The inhibitory effect of DEANO on Ca2+ uptake into mitochondria was confirmed by recording mitochondrial Ca2+ during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. 5 DEANO (10 muM) failed to inhibit Ba2+ entry into TG-stimulated cells when extracellular Ca2+ was buffered below 1 muM, while clear inhibition of Ba2+ entry into store depleted cells was observed when extracellular Ca2+ levels were above 10 muM. Moreover, buffering of intracellular Ca2+ by use of N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA/AM) eliminated inhibition of CCE by NO, indicating that the observed inhibitory effects are based on an intracellular, Ca2+ dependent-regulatory process. 6 Our data demonstrate that NO effectively inhibits CCE cells by cGMP-independent suppression of mitochondrial function. We suggest disruption of local Ca2+ handling by mitochondria as a key mechanism of NO induced suppression of CCE.