We have investigated transcript elongation efficiency by RNA polymerase II on chromatin templates in vitro. Circular plasmid DNAs bearing purified RNA polymerase II transcription complexes were assembled into nucleosomes using purified histones and transient exposure to high salt, followed by dilution and dialysis. This approach resulted in nucleosome assembly beginning immediately downstream of the transcription complexes. RNA polymerases on these nucleosomal templates could extend their 15- or 35-nucleotide nascent RNAs by only about 10 nucleotides in 15 min, even in the presence of elongation factors TFIIF and SII. Efficient transcript elongation did occur upon dissociation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arrested conformation when they encountered nucleosomes. Surprisingly, elongation was no more efficient on nucleosomal templates reconstituted only with H3/H4 tetramers, even in the presence of elongation factors and/or competitor DNA at high concentration. Thus, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.