A new histone-specific acetyltransferase, which is closely associated with nucleosomes prepared from lymphocyte nuclei by treatment with micrococcal nuclease, is described. The acetylating enzyme transfers [3H]acetyl groups from [3H]acetyl-coenzyme A to the endogenous histones H2A, H2B, H3 and H4 in nucleosomes as well as to free histones added to the reaction mixture. Histone H1 is not acetylated by this enzyme. The acetyltransferase was partially purified by DEAE-Sephadex and DNA-cellulose chromatography. The nucleosome-associated enzyme binds to DNA cellulose at low salt concentrations (DNA-binding acetyltransferase), while the previously described histone-specific acetyltransferases have no affinity to DNA under these conditions. This high affinity for DNA may explain the association of DNA-binding acetyltransferase with nucleosomes.