ABSTRACT Restriction maps of an α-amylase structural gene clone, λDm65, and of four putative α-amylase pseudogene clones are presented. Two α-amylase structural genes, inverted with respect to each other, are contained in λDm65. Subregions of internal DNA sequence homology within λDm65 and of cross-homology between the presumptive pseudogene clones and λDm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse α-amylase cDNA clone, pMSa104, were also determined. The presence of functional α-amylase structural genes in λDm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6- kb EcoRI fragments of λDm65 were shown to code for α-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the α-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of λDm65 was detected.