During stroke or head trauma, extracellular K+ concentration increases, which can cause astrocytes to swell. In vitro, such swelling causes astrocytes to release excitatory amino acids, which may contribute to excitotoxicity in vivo. Several putative swelling-activated channels have been identified through which such anionic organic cellular osmolytes can be released. In the present study, we sought to identify the swelling-activated channel(s) responsible for D-[3H]aspartate release from primary cultured astrocytes exposed to either KCl or hypotonic medium. KCl-induced D-[3H]aspartate release was inhibited by the anion channel inhibitors 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), dideoxyforskolin, L-644711, ATP, ITP, 3'-azido-3'-deoxythymidine, DIDS, and tamoxifen but not by cAMP. The cell swelling caused by raised KCl was not inhibited by extracellular ATP or tamoxifen as measured by an electrical impedance method, which suggests that these anion channel inhibitors directly blocked the channel responsible for efflux. Extracellular nucleotides and DIDS, however, had no or only partial effects on D-[3H]aspartate release from cells swollen by hypotonic medium, but such release was inhibited by NPPB, dideoxyforskolin, and tamoxifen. Of the swelling-activated channels so far identified, our data suggest that a volume-sensitive outwardly rectifying channel is responsible for D-[3H]aspartate release from primary cultured astrocytes during raised extracellular K+ and possibly during hypotonic medium-induced release.