A strain of cadmium-resistant Drosophila containing a chromosomal duplication of the metallothionein gene was isolated. This duplication is stably inherited in the absence of selective pressure, and larvae homozygous for it can produce approximately twice as much metallothionein RNA as wild-type larvae. The entire duplication was cloned within a 5.7-kilobase fragment; this fragment contained a direct, tandem repeat of 2.2 kilobases of DNA: 228 bases of 5' flanking DNA, the entire transcription unit, and 1.4 kilobases of 3' flanking sequences. The 3' region of the first repeated unit is joined to the 5' region of the second unit by a 6-base-pair segment we define as the novel joint. This joint forms part of a 10-base-pair inverted repeat of a segment within the 3' region of the first unit. Comparison of the sequences of the 5' and 3' boundaries revealed no extensive regions of similarity at a position corresponding to the novel joint, thus suggesting that a mechanism other than homologous recombination was involved in the origin of this duplication.