The three-helix, ∼65-residue histone fold domain is the most structurally conserved part of the core histones H2A, H2B, H3, and H4. However, it evinces a notable degree of sequence variation within and between histone classes. We used two approaches to characterize sequence variation in these histone folds, toward elucidating their structure/function relationships and evolution. On the one hand we asked how much of the sequence variation seen in structure-based alignments of the folds maintains physicochemical properties at a position, and on the other, whether conservation correlates to structural importance, as measured by the number of residue-to-residue contacts a position makes. Strong physicochemical conservation or correlation of conservation to contacts would support the idea that functional constraints, rather than genetic drift, determines the observed range of variants at a given position. We used an 11-state table of physicochemical properties to classify each position in the core histone fold (CHF) alignments, and a public website (http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/valdar/scorecons_server.pl) to score conservation. We found that, depending on histone class, from 38 to 77% of CHF positions are maximally conserved physicochemically, and that for H2B, H3, and H4 the degree to which a position is conserved correlates positively to the number of contacts made by the residue at that position in the crystal structure of the nucleosome core particle. We also examined the correlation between conservation and the type of contact (e.g., inter- or intrachain, histone-histone, or histone-DNA, etc.). For H2B, H3, and H4 we found a positive correlation between conservation and number of interchain protein contacts. No such correlation or statistical significance was found for DNA or intrachain contacts. This suggests that variations in the CHF sequences could be functionally constrained by requirements to make sufficient interchain histone contacts. We also suggest that inventory of histone residue variants can augment functional studies of histones. An example is presented for histone H3. Proteins 2003;52:454–465. © 2003 Wiley-Liss, Inc.