Enzymatic extracts from a gcn5 mutant and wild-type strains of Saccharomyces cerevisiae were chromatographically fractionated and the histone acetyltransferase activities compared. When free histones were used as substrate, extracts from wild-type cells showed two peaks of activity on histone H3 but extracts from gcn5 mutant cells showed only one. With nucleosomes as substrate, the histone acetyltransferase activities present in extracts from the gcn5 mutant strain were not able to modify H3 whereas wild-type cell extracts acetylated intensely this histone. The activity that acetylated nucleosome-bound H3 behaved as a 170-kDa complex. We suggest that Gcn5p represents a catalytic subunit within a multiprotein complex containing proteins that confer on it the ability to acetylate H3 in nucleosomes.