In Culex pipiens, overproduction of nonspecific esterases is a common mechanism of resistance to organophosphate insecticides. The esterases are attributed to closely linked loci named A and B according to substrate preference, and overproduction of all esterases B is due to gene amplification. Distribution of electrophoretically distinct variants of overproduced esterases A and B is geographically restricted, with the exception of esterases A2 and B2, always found together throughout at least three continents. To determine whether this situation is due to migration or to a high mutation rate, esterase B structural genes and their flanking regions were compared by sequence and/or restriction fragment length polymorphism analysis. Whereas structural genes were similar, flanking regions of electrophoretically dissimilar esterases B varied considerably. In contrast, flanking sequences of esterases B2 from different geographical locations (Africa, Asia, North America) were identical. These results suggest that amplified esterase B2 genes originated from an initial event that has subsequently spread organophosphate insecticide resistance by migration.