In this study the relative importance of adenosine deaminase and adenosine kinase in regulating extracellular adenosine concentration was investigated in rat hippocampal slices labelled with [3H]-adenine. The release of [3H]-purines evoked by electrical stimulation or energy depletion (oxygen and glucose deprivation) was measured and, using high-performance liquid chromatography (HPLC), the proportion of [3H]-label in the form of [3H]-adenosine, [3H]-inosine and [3H]-hypoxanthine was determined. In addition, endogenous purine release was measured by HPLC with UV detection. 10 microM 5-iodotubericidin (5-IT), an inhibitor of adenosine kinase, significantly increased endogenous adenosine release and altered the pattern of [3H]-purine release by increasing the proportion released as [3H]-adenosine, under basal conditions and after electrical stimulation or energy depletion. 5 microM erythro-9-(2-hydroxy-3-nonyl) adenosine (EHNA), an inhibitor of adenosine deaminase, also increased endogenous adenosine release and altered the pattern of [3H]-purine release evoked by energy depletion by decreasing the proportion of [3H]-label released as [3H]-hypoxanthine and [3H]-inosine, whilst approximately doubling that of [3H]-adenosine. In contrast, adenosine release was not altered by EHNA under basal conditions or electrical stimulation. It is concluded that under conditions which provide adequate oxygen and glucose, adenosine kinase plays a much greater role than adenosine deaminase in regulating the extracellular concentration of adenosine. However, adenosine deaminase becomes important in regulating extracellular adenosine concentration when adenosine formation is increased by energy depletion.