Concentration-dependence of multiple actions of phenytoin (PT) on mouse spinal cord neurons in primary dissociated cell culture was studied using intracellular microelectrode recording techniques. At concentrations of 2 to 50 micrograms/ml, PT did not alter resting membrane potential or input resistance. At 1 to 2 micrograms/ml, equivalent to therapeutic cerebrospinal fluid concentrations, PT limited the ability to sustain high-frequency repetitive firing of action potentials during long (500-2000 msec) depolarizing current pulses. There was a progressive reduction of maximal rate of rise (Vmax) of action potentials during the train until firing failed. Recovery of Vmax of single action potentials after repetitive firing was also prolonged. PT did not reduce Vmax of a single action potential at 1 to 2 micrograms/ml, but did so at 3 to 40 micrograms/ml in a voltage-dependent manner. Hyperpolarization partially reversed this reduction of Vmax. Thus, PT may slow recovery of sodium channels from inactivation. At concentrations above 3 micrograms/ml, PT reduced spontaneous neuronal firing with progressive increase in the number of quiescent neurons, reduced calcium-dependent action potential duration and amplitude, eradicated convulsant-induced paroxysmal bursting and augmented postsynaptic responses to iontophoretically applied gamma-aminobutyric acid. Glutamic acid responses were unaffected at PT concentrations of 10 micrograms/ml or less. These actions occurred at concentrations equivalent to toxic cerebrospinal fluid levels in patients and may be related to PT-induced toxicity. We suggest that limitation of sustained high-frequency repetitive firing may account, at least in part, for the anticonvulsant efficacy of PT.