We isolated recombinant genomic DNA clones containing sequences coding for the female-specific vitellogenin proteins of Drosophila grimshawi. By screening with cDNA vitellogenin clones derived from female fat body mRNA we were able to isolate all three genes, namely V1, V2 and V3. The identity of these genes was established first by cell-free translation of the hybrid-selected mRNA followed by protease digestion of the in vitro translation products and second by hybridization of the three genes to electrophoretically separated mRNAs. The transcriptional orientation of these genes was determined. The V1 and V2 genes have opposite orientations with their 5'-ends 1.75 kb apart. S1 analysis demonstrated that the V1 gene has three exons of 310, 400 and 980 bp in length and two introns of about 120 bp. The V2 gene has two exons of 300 and 1260 bp in length and an intron 100 bp long. The V3 gene has three exons of 250, 375 and 820 bp in length and two introns of about 120 bp. The homology, in both sequence and structure, of the vitellogenin genes indicates that they have arisen by duplication events from an ancestral gene. Moreover, the similarity of the V1 and V2 gene positions within the genome of the two distant species D. melanogaster and D. grimshawi suggests a functional coupling of these two genes during vitellogenin gene expression.