H1 histone subfractions exhibit differential abilities in aggregating superhelical DNA, as measured by sedimentation velocity analysis. In 0.15 M NaCl, all the calf thymus H1 subfractions bound to superhelical PM2 DNA to produce nonaggregated H1-DNA complexes as well as rapidly sedimenting, aggregated complexes. Notably, the distribution of the complexes between the nonaggregated and aggregated forms was a function of ionic strength and also depended on which H1 subfraction was complexed to the superhelical DNA. All of the H1 subfractions interacted preferentially with superhelical over relaxed PM2 DNA. The cooperative binding of H1 subfractions to linear T7 DNA produced only aggregated H1-DNA complexes in 0.15 M NaCl, while leaving some DNA free. The compositional and structural variation between the subfractions of H1 histone serves as a basis for their differential effectiveness in H1-induced aggregation of superhelical DNA. The observed sensitivity of aggregation to NaCl concentration is interpreted in terms of a dependence on hydrophobic interactions, such as the proper folding of the particular H1 subfraction and intermolecular interactions between neighboring hydrophobic regions, as well as on nonspecific shielding of DNA charge. These sedimentation velocity analyses augment previous studies on the interactions of H1 subfractions with linear and superhelical DNA using circular dichroism, viscosity, and filter-binding analyses. The involvement of particular H1 and DNA conformations in producing the various types of H1-DNA interactions characterized in these studies might also apply to chromatin structure.