Structural features of the intermediate filaments (IF) of bovine epidermal keratinocytes (keratin filaments) and of baby hamster kidney (BHK-21) cells were studied to define the molecular basis of the similarities and differences in their properties. Purified subunits of the IF were specifically cleaved at their methionine and tryptophan residues. Peptide maps indicated that the IF subunits of BHK-21 cells were similar but not identical to each other and quite different from each of the keratin IF subunits. Intact IF were subjected to limited tryptic digestion, and structurally identical alpha-helix-enriched particles were released. Their properties indicated these IF were composed of a similar three-chain unit which contained regions of coiled-coil alpha-helix interspersed with region of non-alpha-helix. These two types of experiments permitted the construction of subunit domain maps which revealed a common structure: all subunits possessed two alpha-helical domains of the same size that were adjoined by non-alpha-helical domains of variable size. We propose that the reported solubility and immunological differences in these IF and perhaps those of other types of cells are due largely to variations in the size, configuration, and amino acid sequence of the non-alpha-helical regions of the subunits in the IF.