Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam‐binding inhibitor (DBI). The authors have recently shown that β‐amyloid peptide (Aβ) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of Aβ in cultured rat astrocytes. Aβ_25–35 and the N‐formyl peptide receptor (FPR) agonist N‐formyl‐Met‐Leu‐Phe (fMLF) increased the secretion of endozepines in a dose‐dependent manner with EC₅₀ value of ≈2 μM. The stimulatory effects of Aβ_25–35 and the FPR agonists fMLF and N‐formyl‐Met‐Met‐Met (fMMM) on endozepine release were abrogated by the FPR antagonist N‐t‐Boc‐Phe‐Leu‐Phe‐Leu‐Phe. In contrast, Aβ_25–35 increased DBI mRNA expression through a FPR‐independent mechanism. Aβ_25–35 induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of Aβ_25–35 on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that Aβ_25–35 stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC. © 2008 Wiley‐Liss, Inc.