The antitermination protein N from bacteriophage λ (Nλ) interacts with the nut site in its own mRNA, as well as host factors, to facilitate formation of a termination-resistant transcription complex. The conserved, amino-terminal arginine-rich domain of Nλ protein is known to interact with a small RNA hairpin (boxB) derived from the nut site RNA. We have examined the binding of Nλ protein, peptides derived from the amino terminus of Nλ, and the related phage P22 N protein to λ boxB RNAs. To facilitate the study of complexes that are not amenable to gel retardation assays, a new polyacrylamide affinity coelectrophoresis technique (PACE) was developed. Using the PACE assay, we have demonstrated that a 19-amino acid peptide from the amino terminus of Nλ protein binds λ boxB RNA with a Kd,app of 5.2 nM. PACE was also used to study the binding affinity of a number of Nλ peptide and λboxB RNA mutants. The PACE technique is complementary to the traditional gel retardation assay for direct measurement of binding interactions, and will be useful for any procedure that requires a pool of RNAs to be resolved based on their relative affinities for proteins or peptides.