Mammalian spinal cord neurons were grown in dissociated cell culture and used to study the effects of the anticonvulsant barbiturates phenobarbital and mephobarbital, and the anesthetic barbiturates pen-tobarbital, secobarbital, and 1,3-dimethyl-butylethyl barbituric acid on amino acid responses and neuronal membrane properties. All barbiturates augmented responses to GABA and diminished glutamate (GLU) responses, but the anesthetic barbiturates were more potent. The anesthetic barbiturates directly depressed excitability by increasing membrane conductance, an effect reversed by the GABA antagonists picrotoxin and penicillin. Anticonvulsant barbiturates, however, had only minimal GABA-mimetic inhibitory action at high doses. Modulation of synaptic events mediated by GABA and GLU might contribute to barbiturate anticonvulsant activity; and direct GABA-mimetic inhibition, combined with similar modulation of synaptic transmission, might underlie barbiturate anesthesia.