A fusion protein consisting of dihydrofolate reductase and diphtheria toxin A-fragment was made by genetically linking cDNA for the two proteins followed by in vitro transcription and translation in a rabbit reticulocyte lysate system. The dihydrofolate reductase in the fusion protein exhibited enzyme activity and, in the presence of methotrexate which imposes a tight structure on dihydrofolate reductase, it was trypsin resistant, indicating that it was correctly folded. When reconstituted with diphtheria toxin B-fragment, it bound specifically to diphtheria toxin receptors and was translocated into cells upon exposure to low pH. Methotrexate prevented the translocation. Protein synthesis was inhibited in cells incubated with the reconstituted fusion protein, but the inhibition was reduced in the presence of methotrexate. We also made a fusion protein containing a mutated dihydrofolate reductase with much lower affinity to methotrexate. Methotrexate did not prevent translocation of this protein. The data indicate that methotrexate prevents translocation of the fusion protein containing wild-type dihydrofolate reductase by imposing a tight structure on to the enzyme.