Estradiol was measured in rat ovarian venous blood plasma at different stages of pseudopregnancy by radioimmunoassay. Estradiol levels on Days 2 and 3 of pseudopregnancy were less than 50 pcgs/ml plasma. On Day 4 the assay showed a mean peak value of 682 pcgs-/ml at 10:00-12:00 hr. The values then gradually declined. They were 610 pcgs/ml at 14:00-16:00 hr, 467 pcgs/ml at 18:00-20:00 hr, and 225 pcgs/ml at 22:00-24:00 hr. At 10:00-12:00 hr on Day 5, the values were 297 pcgs/ml. There was, however, no significant difference between the values at 22:00-24:00 hr on Day 4 and 10:00-12:00 hr on Day 5. The values determined at various times on Day 4 of pseudopregnancy did not show a linear regression and there was a great variation about the mean. It is therefore suggested that the ovary maintains a plateau of estrogen secretion for some hours on Day 4. This plateau of estrogen secretion probably is of importance in uterine sensitization for decidual cell reaction or implantation. In conclusion, the estrogen surge was found to occur not later than 10:00 hr on Day 4 of pseudopregnancy, rather than during the latter part of the afternoon as has been reported by other workers using biological criteria for estrogen determination.