Acidosis has traditionally been considered to mediate certain types of hypoxic-ischemic injury to the brain. However, the recent demonstration that moderate acidosis will reduce NMDA-mediated currents suggested that acidity could actually protect against types of ischemia and excitotoxicity, and in vitro studies now support this idea. Prompted by this, we have utilized the silicon microphysiometer, a recently-developed instrument that allows for indirect real-time measurement of metabolic rate by detecting proton efflux from small numbers of cultured cells, to determine whether acidity has protective effects upon cellular metabolism. Reducing extracellular pH from 7.4 to as low as 6.0 caused prompt, step-wise, and reversible inhibition of proton efflux rate in cortical and hippocampal cultures both normally and restricted to either glycolysis or oxidative metabolism. Approximately half of the inhibition was due to acidotic effects of NMDA-mediated currents, as demonstrated with NMDA receptor antagonists. Such an inhibition of this indirect metabolic measure could be associated with constant or increased ATP concentrations and represent a beneficial decrease in energy demands upon a neuron. Alternatively, an inhibition of proton efflux rate could be associated with ATP depletion and reflect impaired energy production. We observed a complex interplay between these opposing patterns. Reducing pH to 6.7 for 20 min caused significantly increased ATP concentrations, and prevented excitotoxin-induced ATP depletion. These effects of acidosis involved both NMDA-dependent and- independent actions. More severe (less than pH 6.7) acidosis did not cause ATP concentrations to rise, and if sustained for more than an hour caused a significant decline in ATP concentrations. Thus, despite the recent emphasis on the surprising neuroprotective potential of acidosis, a drop in pH is still likely to have complex and mixed consequences for brain tissue.