[Ca²⁺]_i and oxidative stress (OS) have been measured simultaneously in calf pulmonary artery endothelial cells (CPAE) obtained from the European Collection of Cell Cultures using fura-2 and carboxy-2’,7’-dichlorodihydrofluorescein (C-H₂DCF, the non-fluorescent precursor of C-DCF). In intact cells ATP-stimulates Ca²⁺ release, followed a few seconds later by a transient increase in oxidative stress (OS). Potential sources of reactive oxygen species (ROS) include nitric oxide synthase, mitochondria and NADPH oxidase. Use of the mitochondrial inhibitors rotenone and antimycin A suggested some involvement of mitochondria although the data did not admit to a simple interpretation (Wilkinson & Jacob, 2003).The intracellular localisation of C-DCF in CPAE has been investigated in a permeabilised cell model. CPAE were perfused with a nominally Ca²⁺-free ‘intracellular’ solution (unbuffered [Ca²⁺] = 10-100µM) containing 0.025mg/ml digitonin to permeabilise the plasma membrane. Within about 30 s an increase in [Ca²⁺]_i was seen, concurrently with leakage of cytosolic probe. This was followed a few seconds later by an increase in the C-DCF signal indicating ROS production. C-DCF measurement is in arbitrary units and not ratiometric so the data have been normalised to a mean control response of 100 % for each culture and are presented as mean ± S.E.M. (n = 3 cultures). Analysis has been performed using ANOVA (Minitab GLM).Either chelation of Ca²⁺ by inclusion of 1mM EGTA in the perfusate or inhibition of the mitochondria with the uncoupler FCCP (2µM) prior to and during digitonin application substantially reduced the increase in OS seen following permeabilisation to 18 ± 10 % and 30 ± 12 % of control respectively (both P < 0.05). To further investigate the site of ROS production, cells were co-loaded with the diaceate acetoxymethyl ester of C-H₂DCF (10µM) and the rhodamine-based dye TMRE (10nM) which accumulates in mitochondria. Imaging showed that basal C-DCF fluorescence was non-uniformly distributed. On permeabilisation the cytosolic fraction leaked out leaving a compartmentalised component which was co-localised with the TMRE. These data are consistent with the rise in Ca²⁺ following permeabilisation triggering mitochondrial production of ROS.It has been proposed that in cardiac myocytes 2’,7’-dichlorodihydrofluorescein locates mainly in the mitochondria (Swift & Sarvazyan, 2000). We have shown that this may also be true for the analogue C-DCF in endothelial cells. This has important implications for oxidative stress measurements made using these fluorescein-based probes.