The tetrapeptide AcSer-Asp-Lys-Pro (AcSDKP), a physiological negative regulator of cell proliferation, inhibits the progression of normal quiescent cell to the S phase of the cycle, while it is inactive in the proliferation of permanent cell lines and of freshly isolated leukemic cells. It protects normal hematopoietic stem cells and progenitors from the toxic effects of anticancer drugs. We studied the effects of AcSDKP on the S phase entry of mouse and chicken continuous cell lines MS-K, 3T3, MDCC-PA9, and MDCC-MSB1 lines when they are cultured under these defined conditions. They show that AcSDKP acts on cells previously partially synchronized by culture under conditions of low serum concentrations or serum starvation. Our results demonstrate that AcSDKP reduces the proliferation of these cell of continuous cell lines as it does on hepatocytes or hematopoietic cells in vivo or on freshly isolated cells in vitro, by blocking or retarding their entry into S phase from early G1.