The role of extracellular Ca2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol-bis-(beta-aminoethyl ether) or omission of Ca2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [3H]thymidine into DNA. Addition of calcium to the Ca2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-alpha, the enzyme involved in nuclear DNA replication.