Nucleosomal particles lacking one H2A.H2B dimer interact with RNA polymerase from Escherichia coli more strongly than the complete nucleosomal core particles. Moreover, the in vitro transcription of the H2A.H2B-deficient particles is much more efficient than that of the whole nucleosomal cores, both in the presence and absence of rifampicin. Although a substantial fraction of particles in the preparation of whole nucleosomal cores binds to RNA polymerase, the efficiency of these particles as transcription templates is very small. This block to transcription is partially eliminated when one H2A.H2B dimer is released from the core particle. Our results suggest that the lack of one H2A.H2B dimer from nucleosomal particles might be required for the formation of complexes with RNA polymerase active in transcription.