Nitrocellulose filter binding assays (NCFBAs) have been used for many years to qualitatively and quantitatively determine protein–nucleic acid affinities. While this technique can be robust thermodynamically and fairly simple to perform, the requirement of radiolabeled nucleic acids (typically 32P) has several major drawbacks. Some disadvantages are the short half-life of 32P, the inherent safety concerns, and the cost of working with radioisotopes. Another drawback is that over time the beta emissions cause fragmentation of the nucleic acids. We have modified standard NCFBAs by developing a quantitative nonisotopic chemiluminescent method using biotin-labeled DNA and a dual-filter format. The biotin tag is detected on both nitrocellulose and positively charged nylon membranes by conventional streptavidin-conjugated alkaline phosphatase recognition systems. Quantitation of the photon emissions is simplified by use of a cooled charge-coupled device camera, although exposure to X-ray film and quantitation by densitometry could also be employed. The binding affinity of bacterial manganese superoxide dismutase to nonsequence-specific DNA has been quantitated previously by standard NCFBAs. We have replicated parts of a published binding study using identical solution conditions and the nonisotopic method that we have developed. We provide quantitative agreement between the isotopic and the nonisotopic methods.