A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide ( E B ) and proflavine ( PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured a t the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB. or PF. respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05. and then decreases as r is increased further, mimicking the behavior of' the sedimentation coefficients. viscosities, and gel electrophoresis patterns measured by other workers a t similar values of r. However, L D / A at 520 nm, which is due to DNAhound E B molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of L D / A determined a t 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r , both exhibiting pronounced maxima a t r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another. and in which both are oriented to similar extents in the flow gradient. T h e unexpected differences in the dependencies of (LD/A),,, and (LD/A),2, on r in the case of E B bound to supercoiled DNA, are attributed to differences in the net overall alignment o f t h e E B molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound E B molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound E B molecules. T h e differences in the LD characteristics of the bound P F and E B molecules may be due to subtle differences in the mechanisms of binding, perhaps reflecting differences in the torsional dynamics and local rigidities in superhelical DNA [ Wu e t al. (1988) Biochemistry 27, 8128-81441 induced by these two different intercalating agents.