Incubation of cultured astrocytes in medium without added Ca2+ causes efflux of glutathione, glutamate, taurine, ATP and NAD+. In this study we further investigated the characteristics of this stimulated efflux by HPLC-analysis of purine catabolites including adenosine. Removal of extracellular Ca2+ for 15 min from the incubation medium elicited an increase in extracellular adenosine that was inhibited by several different blockers of gap junctions; carbenoxolone (100 mM) but not by the P2X7 receptor inhibitor Brilliant Blue G (100 nM). The extracellular concentration of adenosine was not blocked by the ectoATPase inhibitor ARL 67156. Thus, the elevated concentration of extracellular adenosine after omission of Ca2+ was caused by increased efflux of adenosine rather than breakdown of extracellular ATP. The implication of the results is that opening of astrocyte channels by omission of extracellular Ca2+ may have protective functions by releasing adenosine in combination with glutathione as we have demonstrated earlier. The finding that adenosine efflux was stimulated by Ca2+ omission and blocked by gap junction blockers indicates efflux via connexin hemichannels. Other channels such as pannexin hemichannels are to our knowledge not gated by extracellular Ca²⁺.