The amplified esterase B gene responsible for resistance to organophosphorus insecticides in Culex mosquitoes is shown to form a DNA "puff" in salivary gland polytene chromosomes. This "puff" is absent in susceptible mosquitoes lacking the amplified gene, and unlike all puffs known today, it is neither related to development nor involved in protein synthesis. The "puff" therefore corresponds to a special arrangement of the very large number of copies (possibly up to 250,000) of the 25-30-kb DNA fragment encompassing the esterase B structural gene in polytene chromosomes. In addition, the amplified and nonamplified esterase B gene(s) were found to be localized on chromosome 2L, and flanked by identical banding patterns, indicating the absence of an important chromosome rearrangement before, during, or after gene amplification.