ABSTRACT—We have investigated the influence of serum and of varying Ca2+concentrations on the DNA synthesis in primary monolayer cultures of adult rat hepatocytes, using a defined basic medium. Supplementation of the medium with 10% horse serum did not significantly affect the time course of the DNA synthesis induced by insulin and epidermal growth factor. Dose effect curves showed that serum moderately sensitized the cells to low concentrations of insulin and slightly sensitized them to epidermal growth factor, but was not required for full responses to maximal concentrations of the hormones. In the serum‐free cultures a wide range of Ca2+concentrations (0.4–1.8 mmol/1) yielded maximal DNA synthesis, suggesting a broad Ca2+optimum of the S phase entry in the hepatocyte monol