Seven lines were isolated with P element insertions in the cytogenetic vicinity of the learning and memory gene, rutabaga, from an enhancer detector screen designed to mark genes preferentially expressed in mushroom bodies. Six of these lines performed poorly in learning and memory tests, and several failed to complement an existing rutabaga allele. Molecular cloning revealed that the P elements were inserted in the putative promoter of the rutabaga gene. RNA in situ hybridization and immunohistochemistry demonstrated that the expression of the rutabaga gene, which encodes a Ca2+/calmodulin-responsive adenylyl cyclase, is markedly elevated in the mushroom bodies of normal flies and that the insertion elements compromised its expression in the new rutabaga mutants. The reisolation of a known learning and memory gene, but with a heretofore unknown expression pattern, strongly supports the postulate that mushroom bodies are principal sites mediating olfactory learning and memory.