Multiple injections of a short acting estrogen, 1,3,5(10)-estratriene-3,16 alpha-diol (16 alpha-E2), have been used to analyze the lag or prereplicative period of approximately 12 h, which precedes the onset of estrogen-stimulated DNA synthesis. A single injection of 1.0 microgram 16 alpha-E2, which itself does not stimulate DNA synthesis, shortened by 3-4 h the lag period between subsequently administered estrogen and the initiation of DNA synthesis. This lag-shortening effect of 16 alpha-E2 was stable for 24 h, but had decayed by 36 h. One or two additional injections of 16 alpha-E2 given sequentially at 3-h intervals each further shortened the lag period but to a lesser extent than the first injection. The results indicate that estrogen induces the accumulation of relatively stable cell changes which are required for the onset of DNA synthesis. The prolonged estrogen requirement during the lag period is not truly discontinuous as previously suggested but rather can be satisfied by discontinuous pulses of estrogen in a ratchet-like fashion because of the stability of their effects.