Fragile X (FraX) syndrome is characterized by mental retardation and a behavioral phenotype that includes stress-related behaviors. Recently, FraX children were shown to have elevated glucocorticoid hormones under basal conditions and an exaggerated hormonal response to stress. In the present study, fragile X mental retardation 1-knockout (Fmr1-KO) and wild-type (WT) mice were subjected to immobilization stress for 30 min or 2 h, killed with paired controls, and the hippocampus, neocortex, and hypothalamic paraventricular nucleus (PVN) assessed by in situ hybridization for effects on c-fos mRNA. The main effect of stress in hippocampus was a reduction in mRNA levels within CA3-CA1 pyramidal cells in both genotypes. Stress significantly reduced CA1 c-fos mRNA in Fmr1-KOs at 30 min (-41%) and 2 h (-57%), whereas in WTs levels were significantly reduced only at 2 h (-57%). In neocortex, 30 min stress significantly increased c-fos mRNA in Fmr1-KOs only (+53%); however, by 2 h levels were reduced in both genotypes versus respective controls. In the paraventricular nucleus, c-fos mRNA levels were significantly, and equally, increased in both genotypes at 30 min. However, at 2 h, mRNA levels were still elevated in the Fmr1-KOs, whereas they had returned to control values in the WTs. Finally, immobilization stress significantly increased serum corticosterone levels in both genotypes at 30 min and 2 h, with Fmr1-KOs exhibiting greater levels than WTs; levels were statistically different at 2 h. These data indicate a greater response to stress in FraX mutants than in WTs, and further support the hypothesis of a dysregulated hypothalamic-pituitary-adrenal (HPA) axis in FraX syndrome.