The mammary cancer cell line CAMA-1 synchronized at the GUS boundary by thymidine block or at the Gl/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17D-estradiol (&), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant & effect on cell proliferation. In synchronized GUS cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90 % mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss & exposure, the response to E2 was again mixed. When & was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The &-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that 1) the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late GI, Gl/S, or G2 are refractory; 2) the E2 stimulation of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, 3) E2 does not facilitate faster cell division; and 4) estrogen-induced cell proliferation or GUS transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated GI events related to tumor growth.