The folic acid analog methotrexate (MTX), a competitive inhibitor of dihydrofolate reductase (DHFR), is used to treat a variety of cancers and autoimmune disorders. However, MTX also causes a wide range of toxic effects in healthy cells and is an established teratogen. Efforts to "rescue" the defects caused by MTX by administering a folate analog or by transgenic expression of a DHFR with an altered affinity for MTX have been attempted in a variety of mammals but limited protection was conferred. As a result, our understanding of the effect of MTX at the molecular genetic level remains incomplete and, in addition, continued mammalian sacrifice is not ideal. Due to the similarity of teratogenic effects produced by MTX in Drosophila melanogaster these insects were transformed with DHFR alleles to determine if rescue could be achieved. The resulting "MTX-resistant" flies were subsequently used to investigate changes in gene expression in response to MTX using semiquantitative reverse transcription PCR. The majority (12/14) of key transcripts that were affected in MTX-exposed females including transcripts involved in cell cycle, defense response, and transport were "rescued" in the "MTX-resistant" transgenic flies. These studies illustrate the utility of this invertebrate model for the investigation of molecular effects of MTX-induced teratogenicity, MTX-resistant DHFRs for gene therapy techniques, and teratogenic protection.