We have isolated four major high mobility group (HMG) proteins designated A, B, C, and D, together with ubiquitin from the ciliate protozoan Tetrahymena. These four HMG proteins are integral structural components of macronuclear nucleosomes. The proteins exhibit solubility properties, chromatographic behavior on carboxymethylcellulose, electrophoretic mobilities on various gel systems, and amino acid compositions similar to those of their mammalian counterparts. HMG-A is the largest, most acidic protein of the group and is phosphorylated in vivo at specific serine residues. HMG-B is both phosphorylated at serine residues and ADP ribosylated. HMG-C is not phosphorylated but is ADP ribosylated. HMG-D, the smallest, most basic protein of the group possesses an unusually high content of serine and threonine residues, and it is highly phosphorylated at both serine and threonine positions in the polypeptide chain.