Decreased pH increases extracellular adenosine in CNS regions as diverse as hippocampus and ventral medulla. However, thus far there is no clear consensus whether the critical pH change is a decrease in intracellular and/or extracellular pH. Previously we showed that a decrease in extracellular pH is necessary and a decrease in intracellular pH alone is not sufficient, to increase extracellular adenosine in an acute hippocampal slice preparation. Here we explored further the role of intracellular pH under different synaptic conditions in the hippocampal slice. When synaptic excitability was increased, either during gamma-aminobutyric acid type A receptor blockade in CA1 or after the induction of persistent bursting in CA3, a decrease in intracellular pH alone was now sufficient to: 1) elevate extracellular adenosine concentration, 2) activate adenosine A1 receptors, 3) decrease excitatory synaptic transmission (CA1), and 4) attenuate burst frequency in an in vitro seizure model (CA3). Hippocampal slices obtained from adenosine A1 receptor knockout mice did not exhibit these pH-mediated effects on synaptic transmission, further confirming the role of adenosine acting at the adenosine A1 receptor. Taken together, these data strengthen and add significantly to the evidence outlining a change in pH as an important stimulus influencing extracellular adenosine. In addition, we identify conditions under which intracellular pH plays a dominant role in regulating extracellular adenosine concentrations.