THE mechanisms that regulate adenosine release in the brain are not well understood. The present study investigated the hypothesis that individual neurons can generate and release sufficient adenosine to regulate their synaptic inputs. We utilized the whole-cell recording technique to apply enzyme inhibitors and nucleotides directly into the cytoplasm of single rat hippocampal CA1 pyramidal neurons. Cytoplasmic delivery of adenosine induced the release of sufficient adenosine to inhibit excitatory synaptic inputs. However, intracellular delivery of nucleotides and enzyme inhibitors failed to increase adenosine receptor-mediated inhibition. These data suggest that while pyramidal neurons in the hippocampus are capable of releasing large amounts of adenosine into the extracellular space, they do not readily form adenosine from endogenous sources.