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There is a large body of evidence that the development of the hypothalamic-pituitary-adrenal (HPA) system in the rat is under maternal regulation. One method used to study the influence of the dam–pup interaction in neonates and weanlings is the separation of mother and litter for 24 h. Previous studies showed that, even at the time of weaning, maternal deprivation results in a dysregulation of the HPA axis at multiple levels. However, the maternal deprivation paradigm usually includes deprivation of food and water, and it was not clear to which extent the observed effects are due to either maternal cues or dehydration and fasting. The primary purpose of the present study was to determine the role of fasting and/or maternal separation on the HPA axis at the time of weaning. Pups at 20 days after parturition are capable of self-feeding and no longer require tactile stimulation to induce eliminative functions. The results indicated that 24 h of fasting led to increased basal levels and further increases in stress induced corticosterone secretion. Fasting also appeared to contribute to the down regulation of basal glucocorticoid receptor mRNA in the hippocampus. In contrast, abrupt weaning irrespective of fasting or dehydration resulted in a suppressed adrenocorticotropin hormone response to an injection of isotonic saline. Although there was an effect of maternal separation on corticotropin-releasing factor mRNA in the paraventricular nucleus, this effect was further exacerbated by the absence of food. Finally, all rats that were separated from their dams showed more efficient negative-feedback. Thus, different aspects of the HPA system appear to respond differentially to either the absence of food or the absence of the mother or both.