Heterotrimeric G(o) is one of the most abundant proteins in the brain, yet relatively little is known of its neural functions in vivo. Here we demonstrate that G(o) signaling is required for the formation of associative memory. In Drosophila melanogaster, pertussis toxin (PTX) is a selective inhibitor of G(o) signaling. The postdevelopmental expression of PTX within mushroom body neurons robustly and reversibly inhibits associative learning. The effect of G(o) inhibition is distributed in both γ- and α/β-lobe mushroom body neurons. However, the expression of PTX in neurons adjacent to the mushroom bodies does not affect memory. PTX expression also does not interact genetically with a rutabaga adenylyl cyclase loss-of-function mutation. Thus, G(o) defines a new signaling pathway required in mushroom body neurons for the formation of associative memory.