The preferred binding sites of echinomycin on DNA can be determined by a method called "footprinting." A 32 P end-labeled restriction fragment from pBR322 DNA is protected by binding to echinomycin, and cleaved by a synthetic DNA cleaving reagent, methidiumpropyl--EDTA⋅Fe(II); the DNA cleavage products are then subjected to high-resolution gel analyses. This method reveals that echinomycin has a binding site size of four base pairs. The strong binding sites for echinomycin contain the central two-base-pair sequence 5′-CG-3′. From an analysis of 15 echinomycin sites on 210 base pairs of DNA, key recognition elements for echinomycin are contained in the sequences (5′-3′) ACGT and TCGT (A, adenine; C, cytosine; G, guanine; T, thymine).