The rate of DNA synthesis was determined in rats with developing and established two-kidney, one clip renal hypertension. Rate of DNA synthesis was measured as [3H]thymidine incorporation into DNA per hour. After stenosis of the renal artery, blood pressure increased over a 2-week period. Five days after clipping, there was an increase in the rate of aortic DNA synthesis before an increase in blood pressure was detected, whereas there was no DNA effect in sham-operated animals. This difference in [3H]thymidine incorporation into aortic DNA could not be accounted for by alterations in thymidine pool sizes. The increase in DNA synthesis was still present 3 weeks after renal artery stenosis, although by that time blood pressure had plateaued. The role of DNA synthesis in the development of renal hypertension was investigated by determining whether inhibition of DNA synthesis with cytosine arabinoside could prevent the increase in blood pressure. Treatment of clipped rats with cytosine arabinoside for 5 days delayed the increase in blood pressure for more than 4 days, as compared with the effect of saline treatment in clipped rats. Although the possibility remains that some effect of cytosine arabinoside other than its effect on DNA synthesis could have influenced blood pressure, there were no differences in body weight, food intake, water intake, or urine output between cytosine arabinoside-treated and saline-treated rats with renal artery clips, and cytosine arabinoside treatment had no effect on blood pressure or body weight in normal rats. These results suggest that an increase in DNA synthesis may be an obligatory step in the genesis of renal hypertension.