The effects of ammonium chloride (3 mM) and beta-methylene-DL-aspartate (BMA; 5 mM) (an inhibitor of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle (MAS] on the metabolism of glutamate and related amino acids were studied in primary cultures of astrocytes and neurons. Both ammonia and BMA inhibited 14CO2 production from [U-14C]- and [1-14C]glutamate by astrocytes and neurons and their effects were partially additive. Acute treatment of astrocytes with ammonia (but not BMA) increased astrocytic glutamine. Acute treatment of astrocytes with ammonia or BMA decreased astrocytic glutamate and aspartate (both are key components of the MAS). Acute treatment of neurons with ammonia decreased neuronal aspartate and glutamine and did not apparently affect the efflux of aspartate from neurons. However, acute BMA treatment of neurons led to decreased neuronal glutamate and glutamine and apparently reduced the efflux of aspartate and glutamine from neurons. The data are consistent with the notion that both ammonia and BMA may inhibit the MAS although BMA may also directly inhibit cellular glutamate uptake. Additionally, these results also suggest that ammonia and BMA exert differential effects on astroglial and neuronal glutamate metabolism.