Initial velocity and product inhibition kinetics of the histone acetyltransferase (EC 2.3.1.48) reaction indicate that the rat liver nuclear enzyme operates under a rapid equilibrium ordered bireactant mechanism. Histone adds first to the enzyme, and under the conditions of the experiment Ka = 0 as acetyl coenzyme A (CoA) concentration approaches saturating conditions. The Km for acetyl-CoA was 2.10 +/- 0.48 micrometer. Inhibition with acetyllysine resulted in a Kiq for the enzyme-acetyllysine complex of 1.96 +/- 0.30 mM. Inhibition with CoA yielded Kip for the ternary complex of 3.19 +/- 0.48 micrometer. These results indicate that the enzyme activity is comparatively independent of histone concentration, and, since the enzyme is sensitive only to acetyl-CoA and CoA concentrations, the enzyme will tend to maintain histones in the acetylated state.