We have isolated the transacting factor PuF that, through its interaction with a nuclease hypersensitive element (NHE) located upstream of the c-myc gene, transactivates the human c-myc gene in vitro (Postel et al., 1989). PuF was recently identified as being encoded by the nonmetastatic 23-H2 (nm23-H2)/nucleoside diphosphate kinase-B (NDPK-B) gene (Postel et al., 1993). In addition to its ability to transactivate the c-myc gene in vitro, PuF/NDPK-B catalyzes the shuttling of gamma-phosphates between nucleoside triphosphates and diphosphates (Gilles et al., 1991; Postel and Ferrone, 1994) and has been postulated to suppress tumor metastasis (Stahl et al., 1991). Here we have extended our studies of PuF and c-myc transcription by testing whether PuF affects c-myc transcription using a transient transfection assay. A plasmid containing the human c-myc promoter-NHE region was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. When cotransfected with a PuF expression vector, CAT activity was elevated 3-4 fold relative to transfections containing the myc-CAT plasmid. In contrast, a myc-CAT reporter plasmid in which the NHE element was deleted showed no increase in CAT activity when cotransfected with the PuF expression vector. From these results we conclude that PuF transactivates the c-myc gene via the nuclease hypersensitive element.