Studies were undertaken to determine the effect of chronic phenytoin exposure on developing neurons. Cerebral cortex from 16‐day fetal mice was utilized to prepare primary dissociated cell cultures. Phenytoin weas added to the cultures 10 days after plating and the cultures were harvested on day 17. Cortical cultures were assayed for neuronal cell number by phase microscopy and for high‐affinity uptake of 3H‐labeled γ‐aminobutyric acid (GABA) by both radioautography and scintillation spectrometry. Neuronal cell counts demonstrated a highly significant decrement in the number of neurons in cultures exposed to phenytoin at 15, 25, and 50 μg/ml. 3H‐GABA‐labeled neurons constituted 13% of the neurons present in both control and phenytoin‐exposed cultures. These data indicate that phenytoin is toxic to cortical neurons in culture and that GABAergic neurons are affected to the same extent as the total neuronal population.