The expression of three unique DNA fragments from Drosophila melanogaster which have been inserted into Escherichia coli (E. coli) via the plasmid, pSC 101, was studied. The hybrid plasmid DNA molecules containing Drosophila DNA were transformed into the minicell producing strain of E. coli, X1411. Drosophila DNA-directed RNA synthesis was studied by hybridizing newly synthesized RNA isolated from the minicells with various DNA fragments which were immobilized on nitrocellulose filters. RNA was synthesized as readily from the inserted Drosophila DNA as from the original bacterial plasmid, pSC 101. In one case, transcription appeared to be initiated preferentially on one of the two strands of a Drosophila DNA fragment regardless of the orientation of that Drosophila DNA fragment with respect to the pSC 101 sequences. Two of the three Drosophila DNA fragments did not induce the synthesis of new polypeptides in minicells as detected by autoradiography of [35S]methionine-labeled polypeptides on polyacrylamide gels. The third Drosophila DNA fragment caused the synthesis of one additional polypeptide of 29 000 daltons. When an 8200 base pair portion of the third inserted Drosophila DNA (63% OF THE TOTAL Drosophila insertion) was removed by digestion with the restriction enzyme, Eco R1, this new polypeptide was no longer synthesized by minicells containing the remaining Drosophila DNA. When the 8200 base pair fragment was placed back into its parent plasmid as an inversion, the new polypeptide did not reappear. In addition, the presence of some, but not all, of the Drosophila DNA insertions affected the relative synthesis of the six polypeptides coded for by the parent plasmid, pSC 101.