The endocannabinoid (eCB) system has recently been implicated in both the pathogenesis of depression and the action of antidepressants. Here, we investigated the effect of acutely or chronically administering antidepressants [imipramine (IMI) (15 mg/kg), escitalopram (ESC) (10 mg/kg), and tianeptine (10 mg/kg)] on the levels of both eCBs [anandamide (AEA) and 2-arachidonoylglycerol (2-AG)] and N-acylethanolamines (NAEs) [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] in various rat brain regions. We also examined the ability of the acute and chronic administration of N-acetylcysteine (NAC) (a mucolytic drug; 100 mg/kg) or URB597 (a fatty acid amide hydrolase inhibitor; 0.3 mg/kg), which have both elicited antidepressant activity in preclinical studies, to affect eCB and NAE levels. Next, we determined whether the observed effects are stable 10 days after the chronic administration of these drugs was halted. We report that the chronic administration of all investigated drugs increased AEA levels in the hippocampus and also increased both AEA and 2-AG levels in the dorsal striatum. NAE levels in limbic regions also increased after treatment with IMI (PEA/OEA), ESC (PEA), and NAC (PEA/OEA). Removing chronic ESC treatment for 10 days affected eCB and NAE levels in the frontal cortex, hippocampus, dorsal striatum, and cerebellum, while a similar tianeptine-free period enhanced accumbal NAE levels. All other drugs maintained their effects after the 10-day washout period. Therefore, the eCB system appears to play a significant role in the mechanism of action of clinically effective and potential antidepressants and may serve as a target for drug design and discovery.