This paper describes an exonuclease-mediated enzyme-linked immunosorbent assay (ELISA)-like assay (EMEA) for detecting the DNA binding activity of nuclear factor kappaB (NF-kappaB). For EMEA, a special double-stranded DNA (dsDNA)-coupled plate was first prepared by immobilizing a DNA probe on an N-oxysuccinimide ester-coated plate. The immobilized DNA probe, which was internally labeled with digoxigenin (DIG)-dT contained a NF-kappaB binding consensus sequence for capturing activated NF- kappaB in analyzed samples. For measurement, the plate was first incubated with a protein sample and then treated with exonuclease III to eliminate the probes not bound by NF-kappaB. Finally, the probes protected by NF-kappaB were colorimetrically detected by an alkaline phosphatase (AP)-conjugated anti-DIG antibody. The major advantage of EMEA is that it detects NF-kappaB without the need for NF-kappaB antibodies. EMEA may provide a general approach for assays of DNA sequence-specific transcription factors for which specific antibodies are unavailable, expensive, or of insufficient quality.