Summary A major histone acetyltransferase was purified from calf thymus chromatin. The apparent K m for acetyl-CoA was found to be 0.5 μM, and that for the DNA-free histones isolated from calf thymus was 0.4 mg/ml. While DNA-free histones isolated from sea urchin sperm were acetylated with an even lower K m (0.08 mg/ml) than the calf histones, the chromatin isolated from sea urchin sperm was not acetylated by this enzyme in vitro. In addition, sea urchin sperm chromatin competitively inhibited the acetylation of DNA-free histories isolated from either calf thymus or sea urchin sperm with a K i of 20 μg/ml. Sea urchin sperm histories are unusual in not being acetylated in vivo ( Easton & Chalkley, 1972). Presumably, this unmodified nucleosomal chromatin binds histone acetyltransferase tightly and inhibits the acetylation, although it is also possible that there may be a specific protein inhibitor in sea urchin sperm chromatin which inhibits the acetylation of histones.