Nitric oxide (NO) and citrulline are produced from L-arginine by the action of NO synthase after activation of excitatory amino acid receptors. In addition to its role in neurodegeneration, there is convincing evidence that NO is also involved in long-term potentiation, a cellular analog of learning and memory in the mammalian nervous system. In the present study, concentrations of L-arginine, citrulline, aspartic acid and glutamic acid were determined in various brain regions of young and old rats. The aim was to examine whether changes in brain concentrations of these amino acids might be indicative of a possible decrease in NO production with ageing, in relation with the well-established decline of cognitive function. Brain aspartic acid, citrulline and L-arginine concentrations were found to be lower in old rats compared to young animals, although the decrease did not always reach statistical significance. In contrast, no change in glutamic acid levels was found. In all brain structures of young and old rats, concentrations of L-arginine were higher than the concentration for NO synthase to function at maximum velocity in the rat brain. Therefore, the decrease in citrulline concentrations found in some brain regions of old rats might be seen, at least partly, as a reflection of a lower production of NO with ageing, although further work is clearly needed to ascertain a decrease in rat brain NO synthase activity with age.