A growing body of evidence suggests the involvement of L-arginine and its metabolites in the ageing and neurodegenerative processes. The present study assessed behavioural performance in 4- (young), 12- (middle-aged) and 24- (aged) month-old male Sprague-Dawley rats, and investigated age-related changes in the activity of two key arginine metabolic enzymes, nitric oxide synthase (NOS) and arginase, and the levels of L-arginine and its downstream metabolites in a number of memory-related brain structures. Aged rats were less anxious and performed poorly in the water maze task relative to the young and middle-aged rats, and both middle-aged and aged rats displayed reduced exploratory activity relative to the young ones. There were significant age-related changes in NOS and arginase activities, and the levels of L-arginine, L-citrulline, L-ornithine, agmatine, putrescine, spermidine, spermine and glutamate, but not γ-aminobutyric acid, in the CA1, CA2/3 and dentate gyrus sub-regions of the hippocampus and the prefrontal, entorhinal, perirhinal, postrhinal and temporal (an auditory cortex) cortices in a region-specific manner. Cluster analyses revealed that the nine related neurochemical variables formed distinct groups, which changed as a function of ageing. Multiple regression analyses revealed a number of significant correlations between the neurochemical and behavioural variables. The present study further supports the involvement of arginine metabolism in the ageing process, and provides further evidence of the effects of animals' behavioural experience on arginine metabolism.