The anticancer antibiotic chromomycin A(3) is a transcription inhibitor which forms two types of complexes with Mg(2+): complex I (1:1 in terms of chromomycin A(3)-Mg(2+)) and complex II (2:1 in terms of chromomycin A(3)-Mg(2+)). These complexes are the DNA-binding ligands. With the broad objective of elucidation of the mechanism for action of this group of transcription inhibitors in eukaryotic systems, we have studied the interaction of the antibiotic with nucleosome core particles under different conditions. We have demonstrated and characterized the role of core histone proteins, particularly the N-terminal tail domains, in the association of nucleosome with both complexes of chromomycin. From a scrutiny of the spectroscopic features of the two bound complexes and comparison of the binding and associated thermodynamic parameters, we have shown the following. Core histone(s) stand(s) in the way of access of the ligand(s) to nucleosomal DNA. N-Terminal intact and chopped core particles interact differentially with the same complex. The modes of interaction of the two complexes, I and II, with the same system are different. Tryptic removal of N-terminal tail domains of core histones enhances the binding potential and access of both complexes of chromomycin to the nucleosomal DNA. Agarose gel electrophoresis of an equilibrium mixture containing either complex I or complex II and a saturating concentration of the core particle has demonstrated that both complexes have a tendency to disrupt the nucleosome structure, leading to a release of nucleosomal DNA. Compared to the N-terminal intact nucleosome, the N-terminal chopped nucleosome is more susceptible to disruption. Therefore, we suggest from the above results that the N-terminal tail domains, which have an important role in eukaryotic gene expression, stand in the way of a free access of external agents such as anticancer drugs to the eukaryotic genome. The significance of the results to understand the molecular basis of the transcription inhibitory capacity of chromomycin is discussed.