ADP-ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4-5-times greater in metaphase cells. 17-22% was in the soluble fraction which contained material released from the cells, 16-22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP-ribosylated proteins corresponding to 78 000, 54 000 and 36 000 Da. In addition metaphase cells contained several other ADP-ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32 000-39 000-Da region suggesting ADP-ribosylation of histone H1 with up to 10 residues of ADP-ribose and in the 17 000-20 000-Da region indicating ADP-ribosylation of core histones. The pattern of ADP-ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP-ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP-ribose) and oligo(ADP-ribose). The results do not lend support to a special function of ADP-ribosylated histones in the mitotic event while certain ADP-ribosylated non-histone proteins may be specific for metaphase cells.