To study the specific membrane actions of the antiepileptic phenobarbital, its effects on frog nerve action potentials were compared with those of pentobarbital. In isolated nerves, the spike amplitude-lowering effect of pentobarbital was augmented by l-epinephrine and blocked by the β-adrenergic blockers sotalol and propranolol, but not by the structurally related local anesthetic d-propranolol or by the α-adrenergic agent phentolamine. These drug interactions, as well as other interactions with calcium and with the dibutyryl derivative of cyclic adenosine monophosphate, suggest that pentobarbital activates axonal β-adrenergic mechanisms. In contrast, the effects of phenobarbital (and procaine) were antagonized by l-epinephrine. Calcium appears to be an important second messenger for the effects of epinephrine on barbiturate action. The effect of phenobarbital differed from that of procaine in not being calcium dependent. These studies indicate that pentobarbital and phenobarbital affect electrically excitable membranes in a specific manner, different from that of nonspecific membrane stabilizers and from each other. In vivo experiments (mouse maximal electroshock seizures) show that the β blockers also interact with pentobarbital, but not with phenobarbital.