Sedimentation and viscosity experiments reveal that when crystal violet binds to closed circular deoxyribo- nucleic acid (DNA) the helix becomes unwound. The average angle of unwinding per bound dye molecule is 9.8 f 0.6'. Viscosity measurements with sonicated rodlike fragments of calf thymus DNA provide evidence that the binding causes an apparent decrease in contour length. Scatchard plots for binding to DNA are curvilinear, concave upward, indicative of strong exclusion effects and/or heterogeneity of binding sites having different affinity. By contrast, binding curves for RNA are curvilinear, concave downward, suggestive of cooperativity. Crystal violet binds preferentially to DNA, its intrinsic asso- ciation constant being 15-fold lower for ribonucleic acid (RNA) at ionic strength 0.01 and 46-fold lower in 0.2 M salt solution. Heat denaturing DNA radically alters the binding mechanism such that Scatchard plots acquire the "humped" appearance characteristic of a cooperative reaction. Its preference for binding to helical DNA is confirmed by a large increase in the "~elting" temperature of calf thymus DNA in buffer of low ionic strength. Raising the ionic strength Studies of the interaction between triphenylmethane dyes and deoxyribonucleic acid (DNA) have produced conflicting accounts of the mechanism of binding (Lerman, 1961, 1964 a,b; Neville & Davies, 1966; Armstrong & Panzer, 1972; Muller & Gautier, 1975). Lerman (1961, 1964a) reported that these dyes increase the viscosity of DNA to an extent comparable to that found for acridines, which led him to propose that both types of ligand intercalate. He also found that on binding to DNA there was a marked decrease in the reactivity of the amino groups of pararosaniline; this he offered as further evidence that the cation is at least partially buried within the helix (Lerman, 1964b). Armstrong & Panzer (1972) confirmed the increase in viscosity up to r (moles of dye bound per mole of DNA phosphate) = 0.1 and suggested that pararosaniline intercalates so as to exclude similar binding at the three nearest-neighbor sites on either side of a bound dye molecule. In addition, they identified a second form of binding at r > 0.125 which did not affect viscosity and which they postulated to involve external attachment to the surface of the DNA helix. In contrast to these reports, X-ray dif- fraction studies on pararosaniline-DNA fibers did not reveal decreases affinity constants generally, and at a salt concen- tration of 0.75 M binding to secondary sites on DNA is sub- stantially reduced. Temperature-jump measurements reveal two fast closely coupled relaxation times at low levels of binding whereas at higher binding ratios an additional two exponential components can be resolved, again closely coupled but well separated from the fast effects. Thus, the ligand forms at least two distinct complexes with DNA at low levels of binding, and there are not less than four bound species at higher binding ratios. The visible absorption spectrum of the crystal violet-DNA complex becomes increasingly batho- chromic and hypochromic as the binding ratio is raised. However, the spectrum of the dye bound to RNA is charac- terized by a strong hypsochromic component and a much greater hypochromism at higher binding ratios than is seen with DNA. These spectral properties are attributed to changes in the propeller-like conformation of the dye due to rotations which affect the relative disposition of the phenyl rings and/or their dimethylamino substitutents.