The neuropharmacological properties of anisatin were tested on the frog spinal cord and the crude synaptic membrane from rat brain. Anisatin (10(-5) M) reduced the amplitude of dorsal root potentials induced by stimulation of the adjacent dorsal root and presynaptic inhibition of the ventral root reflex. Anisatin shifted the dose-response curve for GABA-induced depolarization in the primary afferent terminal to the right and also reduced the maximum response to GABA. [3H]Muscimol binding to the crude synaptic membrane was not inhibited by anisatin. These results indicate that anisatin is a picrotoxin-like, non-competitive GABA-antagonist.