Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-gamma-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.