Lactation was prolonged until 61 days by repeated renewal of litters every week after day 12 in primiparous C3H/He strain mice. On days 12, 19, 40, and 61 of lactation, litters were removed for 5 h and after 1 h of resuckling the synthesis of DNA and RNA in the mammary gland was estimated by the incorporation of (3H)thymidine and (14C)uridine into mammary DNA and RNA in vitro respectively. Mammary nucleic acid content and pituitary and plasma levels of prolactin were also assayed. Nulliparous mice were similarly treated on day 19 of pregnancy. The percentage gain in litter weight per week was highest between days 5 and 12 of lactation, declined until days 26-33 and became steady thereafter. Mammary DNA synthesis was extremely high on day 19 of pregnancy, decreased on day 12 of lactation to less than one-fifteenth of that on day 19 of pregnancy and increased linearly therafter. Changes in mammary DNA content were not so marked, but DNA content was high on days 12 and 19 of lactation. RNA synthesis was highest on day 19 of pregnancy, abruptly decreased on days 12 and 19 of lactation and increased again with the advance of lactation. Mammary RNA content, RNA:DNA and 14C:3H ratios increased from day 19 of pregnancy to days 12-19 of lactation and decreased on days 40 and 61. While the pituitary levels of prolactin were almost constant during lactation, they were significantly higher than those on day 19 of pregnancy. There were only slight differences in plasma prolactin levels at any stage.