In chicken immature erythrocytes, approximately 4% of the modifiable histone lysine sites participate in active acetylation. There are two categories of actively acetylated histone H4. Although both are acetylated at the same rate (t1/2 = 12 min), one is acetylated to the tetraacetylated form and is rapidly deacetylated (class 1), and the other is acetylated to mono- and diacetylated forms and is slowly deacetylated (class 2). We show that the chromatin distribution of the class 1 labeled tetraacetylated H4 species paralleled that of the transcriptionally active DNA sequences. For example, the chromatin fragments of the insoluble nuclear material contained 76% of the active DNA and 74% of the labeled tetraacetylated H4. Class 2 labeled acetylated H4 species were found in repressed chromatin and were enriched in active/competent gene-enriched chromatin fragments. The majority of the histone deacetylase activity (75-80%) was located with the insoluble residual nuclear material. Further, approximately 40-50% of the enzyme activity was associated with nuclear matrices prepared by two methods using high salt and intermediate/high salt extraction. Histone deacetylase was solubilized by extracting the nuclear matrices with high salt and 2-mercaptoethanol, a procedure that generates nuclear pore-lamina complexes. These results demonstrate that histone deacetylase is a component of the internal nuclear matrix.