Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.