Summary The levels of cyclic AMP, DNA, RNA, and protein were measured in morphologically “differentiated” cells induced by noncyclic AMP agents such as 5-bromodeoxyuridine (5-BrdU), 6-thioguanine, serum-free medium (SFM) and X rays. The cyclic AMP levels in 5-BrdU-, SFM-treated cells increased by about twofold, whereas it remained unchanged in X irradiated and 6-thioguanine-treated cells. This shows that X ray and 6-thioguanine probably bypass the step involving the elevation of cyclic AMP. Sodium butyrate increased the cyclic AMP level without causing morphological differentiation, indicating that an elevation of cyclic AMP is not sufficient for the expression of differentiated phenotype. The RNA and protein contents of “differentiated” cells markedly increased. The DNA content of SFM- and 6-thioguanine-treated cells did not significantly change when compared to controls, indicating that the relative distribution of cells throughout the cell cycle was about the same as controls. The DNA content of 5-BrdU treated cells markedly decreased, indicating that most of the cells were accumulated in the G1 phase. The DNA content of X irradiated cells markedly increased, indicating that the expression of differentiated phenotype occurred in the G2 phase and/or in the polyploid cells. This paper was supported by U.S. Public Health Service CA-12247, RR-05357 and DRG-1182 from Damon Runyon Memorial Fund for Cancer Research. We thank Drs. H. Sheppard of Hoffmann-La Roche, Inc., and J. E. Pike of UpJohn Co. for their generous supply of R020-1724 and prostaglandins, respectively. We thank Ms. April Montgomery and Mrs. Marianne Gaschler for their technical help.