► CB1Rs have high density in three layers of the frontal cortex of the rat and mouse. ► CB1Rs are present in frontocortical glutamatergic and serotonergic nerve terminals. ► CB1R activation decreases evoked glutamate release in rat and mouse synaptosomes. ► CB1R activation decreases serotonin release in rat frontocortical synaptosomes. ► Evoked glutamate and serotonin release is greater in the CB1R KO versus WT mice. Both the serotonergic and endocannabinoid systems modulate frontocortical glutamate release; thus they are well positioned to participate in the pathogenesis of psychiatric disorders. With the help of fluorescent and confocal microscopy, we localized the CB1 cannabinoid receptor (CB1R) in VGLUT1- and 2- (i.e. glutamatergic) and serotonin transporter- (i.e. serotonergic) -positive fibers and nerve terminals in the mouse and rat frontal cortex. CB1R activation by the synthetic agonists, WIN55212-2 (1μM) and R-methanandamide (1μM) inhibited the simultaneously measured evoked Ca2+-dependent release of 14Cglutamate and 3Hserotonin from frontocortical nerve terminals of Wistar rats, in a fashion sensitive to the CB1R antagonists, O-2050 (1μM) and LY320135 (5μM). CB1R agonists also inhibited the evoked release of 14Cglutamate in C57BL/6J mice in a reversible fashion upon washout. Interestingly, the evoked release of 14Cglutamate and 3Hserotonin was significantly greater in the CB1R knockout CD-1 mice. Furthermore, CB1R binding experiments revealed similar frontocortical CB1R density in the rat and the CD-1 mouse. Still, the evoked release of 3Hserotonin was modulated by neither CB1R agonists nor antagonists in wild-type CD-1 or C57BL/6J mice. Altogether, this is the first study to demonstrate functional presynaptic CB1Rs in frontocortical glutamatergic and serotonergic terminals, revealing species differences.