Recent studies have revealed that non-Cyclorrhapha insects possess two acetylcholinesterases (AChEs): Drosophila Ace-orthologous (o-Ace) and -paralogous (p-Ace) forms. In these insects, p-Ace is considered as a neural target of insecticides because mutations conferring insecticide insensitivity have been consistently identified in equivalent forms from various insects. To clarify the functional differentiation between the two AChE isoforms, we characterized the expression of two AChE genes in nerve cell-rich and -poor tissues of German cockroaches. The two AChE genes were expressed at comparable levels in both the nerve cord and head. Two AChE isoforms with mutually discriminatory fenitroxon sensitivities were exhibited in the nerve cord, indicating that o-Ace and p-Ace function in the neurons. p-Ace was mainly expressed in the nerve cord and head, while o-Ace was ubiquitously expressed at a comparable level. Moreover, o-Ace transcripts as well as AChE activity were detected in the fat body where no nervous tissue is observed. No AChE activity was detected in the hemolymph. These results show that p-Ace is better as an insecticidal target because of its abundant transcripts in nervous tissue. In addition, the roles of o-Ace, which is present ubiquitously without secretion into the hemolymph in insects, are discussed.