A possible involvement of ADP-ribosylation of chromosomal proteins in the regulation of specific gene expression has been explored. Inhibitors of ADP-ribosylation increased the levels of mRNA for growth hormone and prolactin in cultured GH3 cells and mouse mammary tumor virus (MMTV) in cultured mammary tumor cells. In the latter system endogenous ADP-ribosylation of chromosomal proteins was analyzed. As expected, the inhibitors decreased ADP-ribosylation, but interestingly, glucocorticoids, known regulators of MMTV synthesis, also decreased ADP-ribosylation. This action of the steroids was very rapid; the fall in ADP-ribose was noted within 30 min, the time span in which mRNA synthesis increases. The results show that a decrease in ADP-ribosylation is associated with an increase in mRNA synthesis and that a decrease in endogenous ADP-ribosylation may be an essential step in glucocorticoid activation of specific genes.