Treatment with 25-hydroxycholesterol, an inhibitor of HMG CoA reductase activity, efficiently blocked the proliferation of both human normal mammary epithelial cells (HMEC) and breast cancer cells (MDA231). Upon release of the block there was a rapid increase in the HMG CoA reductase activity, which reached a maximum 4 h after removal of the inhibitor, in both cell types. In MDA231 this increase in enzymatic activity was followed by a rapid initiation of DNA synthesis, whereas this process started considerably later in HMEC. Even in HMEC stimulated to DNA synthesis by insulin, there was first an increase in HMG CoA reductase activity with a maximum 4 h after the addition of the mitogen. If 25-hydroxycholesterol was added along with insulin, the subsequent initiation of DNA synthesis was prevented. Following a 25-hydroxycholesterol treatment as short as 4 h, the onset of DNA synthesis was delayed, indicating that a certain level of HMG CoA reductase activity (= mevalonate synthesis) in the early and mid stage of the prereplicative phase is required for the transduction of the signal leading to initiation of DNA synthesis. In concurrence with these data, it was also demonstrated that giving an exogenous supply of mevalonate to the insulin-stimulated HMEC results in a faster initiation of DNA synthesis.