The deleterious effects of endothelin-1 (ET-1) in the central nervous system (CNS) include disturbance of water homeostasis and blood–brain barrier (BBB) integrity. In the CNS, ischemic injury elicits ET-1 release from astrocytes, behaving through G-protein coupled ET receptors. These considerations raise the question of whether ET-1 influences cellular functions of astrocytes, the major cell type that provides structural and functional support for neurons. Uncontrolled nitric oxide (NO) production has been implicated in sterile brain insults, neuroinflammation, and neurodegenerative diseases, which involve astrocyte activation and neuronal death. However, the detailed mechanisms of ET-1 action related to NO release on rat brain astrocytes (RBA-1) remain unknown. In this study, we demonstrate that exposure of astrocytes to ET-1 results in the inducible nitric oxide synthase (iNOS) up-regulation, NO production, and matrix metalloproteinase-9 (MMP-9) activation in astrocytes. The data obtained with Western blot, reverse transcription-PCR (RT-PCR), and immunofluorescent staining analyses showed that ET-1-induced iNOS expression and NO production were mediated through an ETB-dependent transcriptional activation. Engagement of Gi/o- and Gq-coupled ETB receptors by ET-1 led to activation of c-Src-dependent phosphoinositide 3-kinase (PI3K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK) and then activated transcription factor nuclear factor-κB (NF-κB). The activated NF-κB was translocated into nucleus and thereby promoted iNOS gene transcription. Ultimately, NO production stimulated by ET-1 enhanced the migration of astrocytes through the tyrosine nitration of MMP-9. Taken together, these results suggested that in astrocytes, activation of NF-κB by ETB-dependent c-Src, PI3K/Akt, and p42/p44 MAPK signalings is necessary for ET-1-induced iNOS gene up-regulation. J. Cell. Physiol. 226: 2244–2256, 2011. © 2010 Wiley-Liss, Inc.