Intercalation models for the binding of drugs to DNA involve the postulate of drug-induced local uncoiling of the double helix. Such uncoiling results in removal and reversal of the supercoils of closed circular DNA, revealed by changes in the sedimentation coefficient. The effects of 17 substances on the S 20 of φX174 RF † † Abbreviations used: RF, replicative form; Me-GAG, methylglyoxal- bis-(guanylhydrazone); IDA, irehdiamine A. DNA have been tested, with the object of investigating whether changes in the supercoiling of closed circles may be employed to verify intercalative binding. Ethidium bromide alters the supercoiling in the same fashion previously found with other circular DNA's. Equivalence between the supercoils and the accumulated drug-induced uncoiling of the helix occurs at a binding ratio ν c = 0.04 ± 0.008 ethidium molecules bound per nucleotide, from which the number of superhelical turns in φX174 RF is estimated to be −13.3 ± 2.7. Proflavine, hycanthone, daunomycin, nogalamycin, chloroquine and propidium are all believed to intercalate and all affect the supercoils in the same qualitative fashion as ethidium. Non-intercalating substances tested include spermine, streptomycin, methylglyoxal- bis-(guanylhydrazone), berenil, chromomycin and mithramycin; none appear to remove and reverse the supercoiling. LSD and chlorpromazine are similarly without effect. Irehdiamine A is exceptional; it is believed not to intercalate, yet apparent removal and reversal of supercoiling is found. Possible non-intercalative mechanisms to account for uncoiling of the double helix by irehdiamine A are discussed. Actinomycin D affects the supercoils of φX174 RF in a fashion qualitatively and quantitatively comparable with ethidium, providing support for the intercalation model of Müller & Crothers (1968). Different intercalating drugs yield different values of ν c with φX174 RF. These are used to calculate φ, the apparent unwinding angle per bound drug molecule, based upon φ = 12 ° for ethidium (Fuller & Waring, 1964). For four drugs (proflavine, hycanthone, daunomycin and nogalamycin) the calculated values of φ are significantly less than 12 °, yet 12 ° is supposed to be a minimum for an intercalative process. The discrepancy is attributed to the persistence of a certain proportion of the bound drug molecules in a non-intercalated state. Estimates of α, the fraction in the intercalated state, are presented.