We have previously reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) binds to the pyrimidine-rich strand of the CT element found in the human c-myc gene and activates CT reporter-driven gene expression in vivo. We now characterize the DNA and protein requirements for the interaction of hnRNP K with the CT element. First, hnRNP K is shown to preferentially bind single-stranded DNA over RNA or native double-stranded DNA. Using specific oligoribonucleotide or deoxyribonucleotide probes with specific or nonspecific RNA or DNA competitors, electrophoretic mobility shift assay revealed hnRNP K to be a DNA-binding protein. Specific binding was not simply a reflection of binding to pyrimidine-rich sequences as the number and arrangement of individual CT elements governed interactions with hnRNP K; at least two CT repeats separated by at least three nucleotides are required for binding, indicating the existence of particular stereochemical constraints regulating CT-hnRNP K complex formation. Deletion analysis showed that hnRNP K possesses several nonoverlapping, DNA binding domains, each capable of specific binding with the CT element and preferring DNA over RNA. Each sequence recognition domain is composed of at least one K homology motif, while a larger portion of hnRNP K may be required for stable RNA binding. Additional experiments indicate that the N-terminal 35 residues of hnRNP K are necessary for transactivating the CT element. These results indicate that hnRNP K is a DNA-binding protein and transcriptional activator.