To evaluate estrogen negative feedback in infantile female rats, 9-day-old rats were ovariectomized (OVX) and treated with different doses of estradiol benzoate (Eb, s.c. once daily for 2 days); plasma LH, FSH and estradiol (E2) levels were then determined by RIA. The responses of these rats were compared with those of 25-day-old rats subjected to the same experimental procedures. In intact sham-operated controls, plasma FSH was elevated on days 9--13 and low on days 25--29, whereas LH did not change significantly throughout the period studied. OVX at day 9 or 25 increased the levels of both hormones, with the response faster and larger in the older rats. Treatment with Eb was more effective in suppressing the post-castration rise in gonadotropin levels in 27-day-old than in 11-day-old rats. At day 27 plasma E2 was elevated to pre-castration values by as little as 10 ng of Eb, whereas at day 11 this dose produced plasma E2 titers even higher than pre-castration values. Plasma E2 titers were high at 9--13 days of age and low at days 25--29. OVX on day 9 decreased E2 2--4 days later to about 1/2 the initial concentration, whereas adrenalectomy (ADRX) or ADRX-OVX was followed by an almost complete disappearance of the steroid. When E2 was injected at day 10 in intact rats to elevate plasma E2, E2 remained elevated when measured 3 to 120 min after its injection, but on day 25, 50% of the injected E2 had disappeared from plasma in 90 min. These results provide additional support for the view that estrogen negative feedback, even though present in infantile rats, is less effective than later in life and hence indicate that high gonadotropin titers observed at days 9--13 in the presence of high E2 may be caused by the relative ineffectiveness of the feedback at these early ages. The high E2 titers appear to be caused by an enhanced rate of production of E2 by the adrenals and ovaries and by a reduced metabolic clearance of the steroid.