The glycerol-3-phosphate dehydrogenase (GPDH, E.C. 1.1.1.8) gene of Drosophila melanogaster contains a tandem duplication of a 4.5-kb-long DNA fragment. Survey of the Gpdh gene region by the Southern blot analysis revealed the following features of this gene duplication: (1) The duplication was not observed in chromosome lines that carry In(2L)t, a cosmopolitan chromosomal inversion in this species. The duplication and the inversion are in linkage disequilibrium. (2) The duplication is polymorphic in the Japan and US natural populations examined. Its frequency is 0.26 on an average in In(2L)t-free chromosomes. (3) Triplication is absent or has not become frequent in the populations surveyed. Possible evolutionary factors of this duplication polymorphism are discussed.