A substantial number of patients do not respond sufficiently to antidepressant drugs and are therefore often co-medicated with lithium as an augmentation strategy. Also inhibitors of nitric oxide synthase (NOS) have been used as an augmentation strategy, while inhibitors of NOS exhibit antidepressant-like properties in various animal models. Therefore, we hypothesized that modulation of NOS may be involved in the long-term effects of antidepressants and lithium, and studied the influence of acute and chronic administration of citalopram, alone or in combination with lithium, on NOS activity in hippocampus, cerebellum, and frontal cortex, by determination of L-citrulline being formed. We found that administration of acute or chronic citalopram (5 mg/kg and 20 mg/kg/24h, respectively) alone or in combination with subchronic lithium (60 mmol/kg chow pellet) did not influence the activity of NOS ex vivo in all regions compared to control. In contrast, high doses of lithium caused a significant decrease in NOS activity in vitro. We conclude that basal conditions are unsuitable for the study of antidepressant effects on NOS, and that the neurochemistry of nitric oxide remains unaltered following chronic citalopram or subchronic lithium under normal physiological conditions.