Background and purpose: Previous studies have suggested a regulation of 5‐hydroxytryptamine (5‐HT) neurons by the endocannabinoid system. The aim of our work was to examine the effect of two CB₁receptor antagonists, SR141716A (rimonabant, Sanofi‐Synthélabo Recherche, Montpellier, France) and N‐(piperidin‐1‐yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide (AM251, Tocris Cookson, Bristol, UK), on the firing rate of dorsal raphe nucleus (DRN) neurons.Experimental approach: Single‐unit extracellular recordings were performed to study the effect of CB₁receptor antagonists in slices of the DRN from rat brain.Key results: Rimonabant (1 µM) and AM251 (1 µM) decreased the firing rate of about 50% of all the recorded DRN 5‐HT cells. The GABA_Areceptor antagonist picrotoxin (20 µM) (Sigma) prevented and also reversed the inhibitory effect of rimonabant (1 µM) and AM251 (1 µM), suggesting that CB₁receptors regulate 5‐HT neurons through the GABAergic system. However, the CB₁/CB₂receptor agonist R‐(+)‐[2,3‐dihydro‐5‐methyl‐3‐[(morpholinyl)‐methyl]pyrrolol[1,2,3‐de]‐1,4‐benzoxazinyl]‐(1‐naphthalenyl) methanone mesylate salt (10 µM) (WIN55212‐2, Sigma, St. Louis, MO, USA) failed to change the firing activity of non‐5‐HT (presumably GABAergic) neurons in the DRN. The endocannabinoid N‐(2‐hydroxyethyl)‐5Z,8Z,11Z,14Z‐eicosatetraenamide (anandamide, Tocris Cookson) (10 µM) also inhibited the firing activity of a number of 5‐HT neurons, but this inhibition was not blocked by rimonabant (1 µM) or AM251 (1 µM), and the stable analogue R‐(+) N‐(2‐hydroxy‐1methylethyl)‐5Z,8Z,11Z,14Z‐eicosatetraenamide (methanandamide, Tocris Cookson) (10 µM) did not mimic this effect. The selective CB₁receptor agonist arachidonoyl‐2‐chloroethylamide (ACEA) (1 µM) only slightly increased the firing rate of DRN 5‐HT cells.Conclusions and implications: These results suggest a tonic/constitutive regulation of DRN 5‐HT neurons by the endocannabinoid system, which may occur through a CB₁receptor‐mediated inhibition of the GABAergic system. The inhibitory effect of anandamide may be mediated through a CB₁receptor‐independent mechanism.