In CD1 mice we investigated the hypothalamic-pituitary-adrenal (HPA) axis response to maternal separation for 8 h daily from postnatal d 3 to 5. At d 3 a slow separation-induced corticosterone response developed that peaked after 8 h, and the pups became responsive to stressors. On the second and third day, the response to 8 h separation rapidly attenuated, whereas the response to novelty did not, a pattern reflected by the hypothalamic c-fos mRNA response. If maternal separation and exposure to novelty were combined, then after the third such daily exposure, the sensitivity to the stressor was further enhanced. Meanwhile, basal corticosterone and ACTH levels were persistently suppressed 16 h after pups were reunited with their mothers. To explain the HPA axis desensitization after repeated separation, we found that circulating ghrelin levels increased and glucose levels decreased after all periods of maternal separation, ruling out a role of altered metabolism. Glucocorticoid feedback was not involved either because a glucocorticoid receptor antagonist amplified the corticosterone response after the first but became ineffective after the third separation. In contrast, a mineralocorticoid receptor antagonist decreased and increased corticosterone levels after the first and third period of separation, respectively. In conclusion, the newborn’s HPA axis readily desensitizes to repeated daily maternal separation, but continues to respond to novelty in a manner influenced by a central mineralocorticoid receptor- rather than glucocorticoid receptor-mediated mechanism.