The relative contributions of radioactively labeled fatty acids and glucose to synthesis of glutamate and glutamine were compared in native and dibutyryl cyclic AMP (diBcAMP)-treated primary rat astrocytes. The intracellular specific activities of glutamate and glutamine were 10-fold greater than the specific activities of aspartate or alanine. Butyrate, octanoate and palmitate were equally as effective as precursors for glutamate and glutamine while glucose was 50% as effective as the fatty acids. The specific activity of glutamate and glutamine were identical in the absence of diBcAMP. In diBcAMP treated cells the specific activity of glutamine was greater than that of glutamate when octanoate and palmitate were the labeled precursors. This suggests that cultured astrocytes preferentially utilize free fatty acids for glutamate/glutamine synthesis and that diBcAMP-treated astrocytes contain more than one glutamate compartment.