Bidirectional communication between neurons and glial cells mediated by endocannabinoid signaling has recently been demonstrated. Endocannabinoids released by neurons can activate CB1-Rs on astrocytes, resulting in glutamate release from astrocytes and a consecutive glutamate-mediated modulation of neural functions. It has also been reported that DGL-a and NAPE-PLD, synthesizing enzymes of the two major endocannabinoids, 2-AG and anandamide, respectively, are also expressed by astrocytes. To explore the effect of astrocytic CB1-R activation on 2-AG and/or anandamide release from astrocytes, firstly, we investigated the co-expression of CB1-Rs and DGL-a as well as NAPE-PLD on astrocytes in the superficial spinal dorsal horn by using multiple immunocytochemical staining. It was found that most astrocytic (GFAP-IR) profiles showed positive immunostaining for CB1-Rs and for both enzymes. NAPE-PLD immunoreactive spots were homogeneously distributed around CB1-Rs, whereas the density of DGL-a-IR puncta showed a peak at a distance of 5–6 mm from CB1-Rs, indicating that cytoplasmic Ca²⁺ transients evoked by the activation of CB1-Rs can reach membrane compartments expressing DGL-a and NAPE-PLD. To test the possibility whether 2-AG and/or anandamide released by astrocytes can act on adjacent glial or neural CB1-Rs, we investigated the distribution of CB1-R-IR spots around astrocytic membrane puncta immunoreactive for DGL-a or NAPE-PLD. We found that the density of CB1-R-IR puncta showed a peak at a distance of 5–6 mm from both DGLa and NAPE-PLD immunoreactive spots on astrocytes, indicating that in case of 2-AG and/or anandamide release from astrocytes, the ligands may act on both neural and glial CB1-Rs. In order to test these possibilities in functional studies we established a primary astrocyte culture. Since we found that cultured spinal astrocytes also express CB1-Rs and both DGLa and NAPE-PLD, this in vitro system can give us a splendid opportunity to investigate the possible mechanisms of endogenous cannabinoid release from astrocytes.