The antigenicity and composition of chromatins differ markedly in chromatin preparations obtained by different procedures. Rat Novikoff hepatoma chromatin (NC) obtained by the "salt precipitation" and the micrococcal nuclease digestion procedures using significant levels of EDTA and NaCl each shows a common complement fixation (CF) capacity, exceeding chromatin preparations obtained from normal rat liver when tested with rabbit antisera raised to dehistonized NC. In contrast, "structured" NC preparations, which have been postulated to retain a native physical conformation, show minimal CF capacity when tested with the same antiserum but show high CF following elution of histones. While further progressive elution of non-histone proteins (NHPs) did not alter the CF capacity per unit DNA, the completely separated DNA and NHP fractions each showed minimal CF. The data suggest that the antigens detected in the CF assay predominantly represent an artifactual but specific complex of DNA and NHP arising from a denaturation of the native chromatin following elution of metal ions or histones. A qualitatively similar profile of NHPs in salt-precipitated NCs shows a range of total protein/DNA ratios, suggesting that the NHPs found in chromatin preparations may not be intrinsic to the native chromatin structure.