A dramatic, time-dependent loss of L-glutamine was observed in mouse and rat hippocampal slices equilibrated in normal artificial CSF under static (no-flow) and superfused (constant-flow) conditions. Concomitant with the decline in L-glutamine, there was a significant, but less pronounced, decrease in levels of the neurotransmitter amino acids, gamma-aminobutyric acid, L-aspartate, and L-glutamate. The disappearance of L-glutamine was a result of diffusion from the tissue to the artificial CSF rather than chemical or biochemical transformation. The loss of amino acids from the hippocampal slices was prevented to different degrees by the addition of 0.5 mM exogenous L-glutamine to the artificial CSF. The levels of newly synthesized amino acids were also determined, because they may be more indicative of the neuronal activity than the total tissue levels of amino acids. The effects of perturbations in glutamine (length of the equilibration time and addition of exogenous glutamine) on newly synthesized glutamate were more pronounced under 4-aminopyridine-stimulated than control (unstimulated) conditions. Therefore, a loss of L-glutamine from the hippocampal slices may have neurophysiological effects and warrants further investigation.