Objective(s):Some pathologic situations such as diabetes and metabolic syndrome are associated with alternation in nitric oxide level. Incidence of these condition increases with aging. On the other hand, insulin secretion is modulated by nitric oxide, and nitric oxide synthase (NOS) activity is also altered in diabetes. In this study, modification in the enzyme activity associated with aging and also optimized procedure for islet NOS assay was investigated. Materials and Methods: Male Wistar rats were randomly divided in two experimental groups: A: adult rats; were 4 month old and B: old rats; were 12 month old. In all groups, plasma glucose, insulin and NOX (nitrite + nitrate = NOX) were measured, and also insulin secretion in isolated pancreatic islet with or without L-NAME was investigated. Furthermore, the inducible NOS activity with L-citrulline measurement in islets was measured. Results: L-citrulline was quantified using one step HPLC column. Aging induced hyperglycemia (P