To evaluate the response of astrocytes in the auditory pathway to increased neuronal signaling elicited by acoustic stimulation, conscious rats were presented with a unilateral broadband click stimulus and functional activation was assessed by quantitative autoradiography using three tracers to pulse label different metabolic pools in brain: [2-14C]acetate labels the 'small' (astrocytic) glutamate pool, [1-14C]hydroxybutyrate labels the 'large' glutamate pool, and [14C]deoxyglucose, reflects overall glucose utilization (CMR(glc)) in all brain cells. CMR(glc) rose during brain activation, and increased activity of the oxidative pathway in working astrocytes during acoustic stimulation was registered with [2-14C]acetate. In contrast, the stimulation-induced increase in metabolic activity was not reflected by greater trapping of products of [1-14C]hydroxybutyrate. The [2-14C]acetate uptake coefficient in the inferior colliculus and lateral lemniscus during acoustic stimulation was 15% and 18% (p < 0.01) higher in the activated compared to contralateral hemisphere, whereas CMR(glc) in these structures rose by 66% (p < 0.01) and 42% (p < 0.05), respectively. Calculated rates of brain utilization of blood-borne acetate (CMR(acetate)) are about 15-25% of total CMR(glc) in non-stimulated tissue and 10-20% of CMR(glc) in acoustically activated structures; they range from 28 to 115% of estimated rates of glucose oxidation in astrocytes. The rise in acetate utilization during acoustic stimulation is modest compared to total CMR(glc), but astrocytic oxidative metabolism of 'minor' substrates present in blood can make a significant contribution to the overall energetics of astrocytes and astrocyte-neuron interactions in working brain.