Circadian rhythms of behaviour and gene expression are coupled to endogenous neuronal oscillators located in the hypothalamic suprachiasmatic nuclei (SCN), which are synchronised by the environmental light cycle. Besides light, other factors such as the pineal hormone melatonin, temperature and feeding have entraining properties. During senescence, the circadian system becomes weaker and susceptible to desynchronisation. It is unknown to what extent age-related changes are the result of the deterioration of the hypothalamic master clock. Supplementing ageing mice with melatonin as well as maintaining them on a hypocaloric diet extends the life span and delays age-related diseases. By means of DNA microarrays and the quantitative polymerase chain reaction, we have conducted an exploratory study to compare the effect of long-term melatonin substitution (MEL) and caloric restriction (CR) on circadian gene expression in hypothalamic samples, which contained the SCN as well as other important nuclei involved in nutrient balance, reproduction, and so on. Over 4% of the hypothalamic transcripts showed an overt circadian rhythm in expression, and many of these contain E boxes in their promoter regions, suggesting a direct regulation by circadian clock genes. MEL and CR significantly influenced some of these rhythmically expressed transcripts, but often in opposite ways. Importantly, our studies emphasise that the apparent direction of treatment effects (i.e. up-regulation versus down-regulation) depends on the time of day at which the samples are compared.