Abstract MicroRNAs (miRNAs) are small non-coding RNAs that control gene expression during cellular processes such as differentiation and development. Alterations in miRNA expression have been linked to age-related changes in mouse liver and Alzheimer disease in the human brain. We decided to investigate a possible role for miRNA activity in age-related thymic decline. To address this question, we isolated lin- triple negative (TN) thymocytes from young (4 months) and aged (24 months) mice, and compared the miRNA profile of 625 miRNAs using qPCR. Thirty-two percent of the miRNAs (199) exhibited a 3-fold or greater increase in the aged animals while only 5% (34 miRNAs) were down-regulated by 3-fold or more. The magnitude of the differences in miRNA expression between young and aged thymocytes was unexpected. The TN population encompasses at least four identifiable subpopulations, representing distinct steps in differentiation. It is likely that these subpopulations differ in miRNA profile. These TN populations are certainly present in varying frequency in young and aged murine thymus. Therefore, some of the overall miRNA expression differences may reflect compositional differences in the TN populations used for RNA isolation. This possibility is being examined by analysis of individually sorted subsets. More intriguing is the possibility that the large number of highly expressed miRNAs found in the aged TN population is indicative that protein expression in these cells is rigidly controlled. We are investigating this possibility by identifying gene targets for a subset of the most highly expressed mRNA. These data provide us with essential information concerning miRNA expression changes that may be associated with the age-specific decline in thymopoiesis.