Abstract The mechanisms by which GH facilitates the recovery of thymopoiesis during age-associated thymic atrophy are unknown. Herein, we analyzed the age-related lipid alterations in total thymus and thymocyte preparations derived from 04 to 18 month-old mice, and in thymi of GH-treated aged mice. Electrospray tandem mass spectrometry permitted us to detect increased amounts of tryglicerides (TG), free-cholesterol and C24:1 sphingomyelin in the total thymus of old mice as compared to those of young. Moreover, we verified a decrease in the sulfatide C24:1 and in unknown long-chain ceramides, and an increase in 4-HNE and 8-epi-prostaglandin F2α (PGF) in the thymi of older animals. Using immunohistochemistry, we verified an increase in the expression of TNFα and CD204 within the aged thymus sections. In analyzing thymocytes, we found a decrease in TG, free cholesterol and in sphingomyelin species with age. Interestingly, we detected a significant amount of ceramide C24:1 in thymocytes derived from old mice but not young. Following GH treatment, we observed a significant decrease in TG, free-cholesterol and PGF in the thymus of older animals. GH treatment significantly increased the amount of dihydroceramides and promoted a trend to decrease 4-HNE levels compared to thymi of age-matched denatured GH-treated control mice. These results suggest that increased oxidative stress associated with perturbed lipid metabolism within aged thymus can be attenuated after GH treatment.