Calf pulmonary endothelial cells (CPAE, obtained from the European Collection of Cell Cultures) respond to ATP with a biphasic increase in cytosolic Ca²⁺ ([Ca²⁺]_i) and a transient production of reactive oxygen species (ROS), detected using fura-2 and carboxy-2′,7′-dichlorodihydrofluorescein (C-DCF), the latter being a probe that is sensitive to H₂O₂. Results obtained using the mitochondrial inhibitors, antimycin A (AA), rotenone (ROT) and FCCP in intact CPAE, and FCCP in permeabilised CPAE implicated mitochondria as the source of the ROS (Wilkinson & Jacob, 2003, 2004) . Our hypothesis is that the increase in cytosolic [Ca²⁺]_i stimulates the mitochondrial electron transport chain, resulting in ROS production. We measured either the transient increase in C-DCF fluorescence in intact CPAE in response to 100mM ATP or the maximum rate of increase of C-DCF fluorescence in CPAE permeabilised with 50μg/ml digitonin in an ‘intracellular’ solution of (mM) Na⁺ 10, K⁺ 135, Mg²⁺ 1, Cl^– 147, HEPES 20, and a nominal 100μM Ca²⁺. This dose of digitonin selectively permeabilises the plasma membrane without releasing C-DCF trapped in the mitochondria. Data were analysed using a mixed model analysis (SAS) of the logarithms of experimental values. P values for comparison with control were calculated using Dunnett’s test. Effects are quoted as a percentage, with the error estimated from the SEM of the logarithmic values. The n values quote the number of cultures; within each culture a minimum of 4 coverslips were measured. Testing the effect of AA on intact CPAE in an additional culture revealed that the potentiation of the C-DCF response previously reported as insignificant was significant (41±18%, P<0.01, n=3). ROT and AA correspondingly stimulated the C-DCF response in permeabilised CPAE by 66±30% (P=0.027, n=3) and 93±48% (P=0.035, n=2) respectively. The NADPH oxidase is generally recognised as a major source of ROS production in endothelial cells but its inhibitor apocynin (50mM in 0.01% DMSO) caused only a small, insignificant attenuation (13±23%, n=2) in the ATP response of intact cells. Both these experiments provide data confirming our hypothesis that the source of the ROS is mitochondrial. We tested the effect of inhibiting nitric oxide synthase (NOS) using 10μM L-NIO. In intact CPAE, L-NIO significantly potentiated the response (68±31%, P=0.01, n=2) but in permeabilised cells its effect was insignificant (10±22%, n=2). NO could be reducing the C-DCF signal by combining with superoxide to form peroxynitrite, preventing its dismutation to H₂O₂. Furthermore, the source of the NO appears to be cytosolic since the effect of L-NIO was not seen permeabilised in cells.