Avermectin B1a (AVM B1a), a widely used insecticide and acaricide, is reported to both activate and inhibit gamma-aminobutyric acidA (GABA(A)) receptor function in mammalian brain. This study attempts to resolve these seemingly contradictory results by examining the binding properties of AVM B1a and its effects on the GABA-gated chloride channel with primary cultures of rat cerebellar granule neurons as a model system. Specific binding of [3H]AVM B1a in intact neuron cultures is time- and concentration-dependent and is displaceable by AVM analogs. Scatchard analysis of [3H]AVM B1a binding reveals high- and low-affinity sites with K(D) values of 5 and 815 nM, respectively. AVM B1a alters the binding of [3H]ethynylbicycloorthobenzoate at the noncompetitive blocker site in a biphasic manner; activation is evident with 10 to 300 nM AVM B1a after 5 to 10 min incubation and inhibition with an IC50 of 866 nM after 60 min incubation. Consistent with this observation, 36Cl- influx is stimulated by AVM B1a at 3 to 100 nM and inhibited at 1 to 3 microM. GABA-stimulated 36Cl- influx is completely blocked by both [3H]ethynylbicycloorthobenzoate and 12-ketoendrin (two GABA-gated chloride channel blockers) and AVM B1a at 1 to 1.5 microM. Also, 36Cl- influx induced by AVM B1a at 10 nM is suppressed by the two channel blockers. Thus, AVM B1a binds to two different sites in the GABA-gated chloride channel with dual effects, i.e., activating the channel on binding to the high-affinity site and blocking it on further binding to the low-affinity site.