Release of glutathione (GSH) from astrocytes is essential for the supply of neurons with the GSH precursor cysteine. In order to test whether gap junction hemichannels could contribute to GSH release from astrocytes, we incubated astrocyte-rich primary cultures from neonatal rat brain in the absence of divalent cations, a condition that is known to increase the opening probability of hemichannels. During incubation in divalent cation free incubation solution (DCFS) the cells remained viable and released about 50% of the initial cellular GSH within 15 min. This extracellular GSH accumulation in DCFS was lowered by the presence of Ca2+ in a concentration dependent manner with a half-maximal inhibition at a Ca2+ concentration of 107+/-46 microM. Extracellular GSH accumulation in DCFS was also blocked by the divalent cations Mg2+, Ba2+ and Sr2+ as well as by the known gap junction inhibitors carbenoxolone (CBX), flufenamic acid (FFA) and lanthanum chloride. In contrast, the P2X7 receptor blocker brilliant blue G (BBG) did not affect GSH release in divalent cation free solution. This pharmacological profile strongly suggests that astrocytes are able to release GSH via open hemichannels. This release of GSH may have severe consequences for the antioxidative defense and for the GSH homeostasis in pathological brain.