A flow cytometric study was carried out on the effects of tumor-promoting 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-8) M) on HeLa cells synchronized by amethopterin for DNA synthesis. Cells treated with TPA at the time of release from the amethopterin block showed a delayed passage through S phase and partially through G2 in their immediate life span as measured 24 hr after release. This late G2 delay was not observed when TPA was added to cells during late S or G2 phase. In this case, however, a direct inhibition of cells in G2 became evident as observed about 15 hr after release from block. None of these effects was caused by the nonpromoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10(-6) M). These data support observations obtained with asynchronous cultures. The TPA effects resemble those reported after X-irradiation of cell cultures.