Experiments were carried out to characterize the prolactin stimulation of labeled uridine uptake into mammary gland explants which were initially preincubated for 2 days in medium containing insulin plus hydrocortisone. Prolactin was found to enhance labeled uridine uptake after 4 h but not after 2 h when uridine concentrations ranging between 0.1 muM and 1 mM were tested. The effect of prolactin on labeled uridine uptake also appears to require ongoing RNA and protein synthesis since incubation with a variety of inhibitors of RNA and protein synthesis abolished the prolactin effect. It was further discovered that prolactin stimulates the labeling of the cellular pools of the phosphorylated derivatives of uridine while the quantity of label present in the uridine and uracil pools was not affected. It is thus possible that the effect of prolactin on labeled uridine uptake may be caused by the enhanced phosphorylation of the uridine. It was also found that the onset of the effect of prolactin on the phosphorylation of uridine to UTP was not different from the onset of the effect of this hormone on labeled uridine uptake and its incorporation into RNA. Additional studies were carried out to determine if the effect of prolactin on RNA synthesis could be dissociated from its effect on the uptake of precursor substances. For this purpose the effect of prolactin on the uptake of radiolabeled phosphate and its incorporation into RNA was studied. Prolactin stimulated the incorporation of phosphate into RNA after 4 h but the uptake of phosphate was not affected by the hormone at that time. Further, the effect of prolactin on phosphate incorporation into RNA was temporally identical to the hormonal effect on the incorporation of 3H-uridine into RNA.