The actions of PRL on RNA and casein synthesis were tested in mouse mammary gland explants that were cultured with medium containing several concentrations of calcium ions. The PRL effects on both RNA and casein synthesis were the same when calcium concentrations between 0.3-11.3 mM were employed. In the absence of extracellular calcium ions, however, PRL had no action on RNA or casein synthesis, but a significant stimulation of ornithine decarboxylase activity by PRL was observed in the absence of calcium. Stimulatory actions of insulin, cGMP, and prostaglandin F2cc on RNA synthesis in the cultured explants were observed in the absence of calcium, but arachidonic acid had no effect under these conditions. It is concluded that specific actions of PRL in the mammary gland require the presence of extracellular calcium ions. (Endocrinology106: 1360, 1980)