Cultured astrocytes, activated to express the inducible form of nitric oxide synthase, produced up to 1 μM nitric oxide (NO) measured by a NO-selective electrode, while non-activated cells produced no detectable NO. The production of NO was associated with an inhibition of cellular respiration, measured simultaneously by an oxygen electrode. The inhibition of respiration was rapidly reversed by inhibiting the NO synthase or by binding the NO with haemoglobin. The respiratory inhibition had an NO, oxygen and substrate dependence consistent with NO-inhibition at cytochrome oxidase. This is the first demonstration that cells can reversibly inhibit mitochondrial respiration via NO production. This inhibition is large and potentially important in a range of pathophysiological conditions.