The dissociation of daunomycin from DNA was examined using a sodium dodecyl sulfate-sequestered stopped flow procedure. Two dissociation processes were observed with calf thymus and bacterial DNA, with approximately 45% of the amplitude associated with the faster process. Both processes were largely independent of DNA sequence for bacterial DNA, comprising 30-70% (G + C) content. The rate of both processes increased by a similar amount with increasing ionic strength. The faster process decreased with decreasing drug loading, whereas the slower process was independent of drug loading. Only one dissociation event was observed for the dissociation of daunomycin from four different synthetic polydeoxynucleotides. All observations are consistent with a parallel model of sodium dodecyl sulfate-induced dissociation of daunomycin from DNA, where the two processes observed reflect two resolvable processes that may be comprised of a series of rate constants for the dissociation of drug from differing environments. The slower process observed with bacterial DNA (0.5-0.7 sec-1) is related to dissociation from preferential 5'-CA DNA-binding sites, whereas the faster process reflects dissociation of drug from lower affinity sites on heterogenous DNA (3.2-4.1 sec-1). Dissociation of daunomycin from four different synthetic polydeoxynucleotides (which did not contain the 5'-CA preferential daunomycin-binding site) exhibited dissociation rates characteristic of low affinity sites (3.3-4.8 sec-1).