The CT element of the c-myc gene is required for promoter P1 usage and can drive expression of a heterologous promoter. Both double strand (Sp1) and single strand (hnRNP K) CT-binding proteins have been implicated as mediators of CT action. Although significant levels of CT activity persisted following Sp1 immunodepletion, EGTA totally abolished transactivation, thus implicating another metal requiring factor in CT element activity. As hnRNP K binds to one strand of the CT element, but has no metal requirement, the opposite (purine-rich strand) was examined as a target for a metal-dependent protein. A zinc-requiring purine strand binding activity was identified as cellular nucleic acid binding protein (CNBP), a protein previously implicated in the regulation of sterol responsive genes. Two forms of CNBP differed in their relative binding to the CT- or sterol-response elements. CNBP was shown to be a bona fide regulator of the CT element by cotransfection of a CNBP expression vector that stimulated expression of a CT-driven but not an AP1-dependent reporter. These data suggest that hnRNP K and CNBP bind to opposite strands and co-regulate the CT element.