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The effect of ovarian hormones on DNA polymerase activity in the mouse mammary gland was determined. The rate of [³H]thymidine triphosphate incorporation into DNA during incubation in vitro was used as a measure of DNA polymerase activity in the post-microsomal supernatant fraction of the mammary tissue. A negligible amount of DNA polymerase activity was present in the mammary tissue of ovariectomized mice which had no hormone treatment. Daily subcutaneous injections of 1 µg oestradiol-17β plus 1 mg progesterone induced a sixfold increase of DNA polymerase activity in the mammary tissue after two injections. A single intraperitoneal injection of the same dose also induced a similar rise of DNA polymerase activity and the effect was evident within hours after the treatment. The results provide evidence that stimulation of cellular biosynthesis by the growth-promoting ovarian hormones is associated with the activation of a specific enzyme involved in DNA replication.