Brain tissue is extremely sensitive to a reduction in blood flow (cerebral ischaemia) and even brief ischaemic episodes can produce irreversible neurological deficit. Experimental studies of cerebral ischaemia have identified several dominant processes which alone or combined underlie ischaemia-induced neuronal damage. Among these, tissue acidosis is a critical factor. This study focused on acid-base changes in the extracellular fluid, the neuronal microenvironment, associated with both ischaemia and spreading depression. The latter phenomenon, which occurs in the region surrounding the ischaemic core associated with focal ischaemia, produces a transient disruption in ionic gradients, and as with ischaemia, leads to failure of residual acid-base homeostasis with subsequent acidification of brain tissue. By investigation of the changes in extracellular concentrations of carbonate, hydrogen ions and lactate, especially in relation to the occurrence of depolarisation, this study has demonstrated the following: (i) The transmembrane mechanisms contributing to intracellular acid-base regulation are markedly activated during ischaemia, resulting in a rapid acidification of the extracellular fluid. (ii) The efficacy of these mechanisms is abolished as the cellular membrane permeability to ions, including H+ and pH-changing anions, suddenly increases with depolarisation. (iii) Various cells of the central nervous system may have a different acid-base homeostasis, at least with regard to membrane permeability and ionic exchange mechanisms for HCO3/CO32. (iv) Efflux of intracellular lactate is markedly reduced when cell membrane ionic gradients are perturbed, confirming the involvement of a transporter. (v) Probenecid, used to block lactate transport across the cellular membrane, inhibited K+-induced spreading depression and reduced the amplitude of anoxic depolarisation. These data strengthen the concept that anoxic depolarisation and spreading depression are critical events in the pathophysiology of cerebral ischaemia and other related insults and therefore, relevant targets for neuroprotective strategies against stroke.