High brain concentration of ammonia (NH4+/NH3) results in deleterious effects on the central nervous system and is a key factor in the pathogenesis of hepatic encephalopathy. We have previously shown that an increase in NH4+/NH3 concentra- tion results in a transient increase in [Ca2+]i in cultured astrocytes. This response is linked to the NH4/NH3-induced alkalinization and results from Ca2+ release from intracellular stores (Rose et al., 2005). Here we report that also the removal of NH4+/NH3 after 10min incubation triggers a Ca2+ increase, but this response is insensitive to thapsigargin indicating that it was not released from endoplasmic reticulum stores. The Ca2+ response was not affected by removal of extracellular [Ca2+], but blocked in the presence of CCCP, which interferes with the uptake of calcium ions by mitochon- dria indicating that NH4+/NH3 removal triggers Ca2+ release from mitochondria. We have two lines of evidence that this [Ca2+]i increase is not linked to the cytoplasmic acidic shift which results from removal of NH4+/NH3: (1) The weak acid propionate (PA+/PA) triggers first an acidic shift and subsequently an alkalinization, but only the alkalinization resulted in an increase in [Ca2+]i . (2) We mimicked the pH response of NH4+/NH3 application and removal by first applying the weak base TMA+/TMA and subsequently PA+/PA. Only the alkaline shift resulted in a Ca2+ transient, but not the acid shift. We therefore conclude that NH4+/NH3 acts on mitochondria independent of its effect on cytoplasmic pH.