Analysis of acetylation in the two histone H3 variants of alfalfa by acid/urea/Triton-polyacrylamide gel electrophoresis has established that the minor variant H3.2 has a 2-fold higher level of acetylation than the major variant H3.1. Purification and sequence analysis of both variants showed sequence identity across the complete amino-terminal domain, which contains the 6 modified lysines 4, 8, 14, 18, 23, and 27. The two proteins have different distributions for acetylation: mono-, di-, and tri-methylation. The higher level of acetylation of H3.2 was confirmed in a wider pattern across all 6 lysines. Lysine modification levels varied for all sites in both proteins between 5 and 95%, with combinations of one to four types of modification co-existing at each residue. Additional sequence analysis of the H3.1 and H3.2 proteins and of tryptic core peptides established that the two histones differ only in residues 31, 41, 87, and 90. This indicates that major histone H3.1 is the product of the major alfalfa histone H3 gene and makes it likely that H3.2 is the product of the minor H3 gene, known from a partial cDNA clone. The variant-specific differences in lysine modifications in protein domains with identical primary structures suggest that the pattern and level of lysine modifications may be directed by the distinct chromatin environments of the two histone H3 variants.