We report that NO synthase activity, as expressed by citrulline and nitrite formation in brain homogenates, is decreased in 24-month old in comparison to 3-month old rats. In particular, a Ca(++)-dependent NO synthase activity was detected in homogenates obtained from cortical, hippocampal, cerebellar and lower brain stem slices from both 3- and 24 month-old rats. The amount of citrulline generated from L-arginine was significantly decreased in the hippocampus and lower brain stem by 40 and 48%, respectively. No changes were observed in NO synthase activity in cortical and cerebellar homogenates. Thus, the L-arginine-NO pathway seems to be impaired in selected areas of rat brain and this may contribute to the understanding of pathophysiological mechanisms underlying age-related cerebral disorders.