1. A method of acid ethanol extraction and gel filtration was used to obtain a ;luteinizing hormone (LH)-free' fraction of blood collected from the cut pituitary stalk of rats (termed hypophysial portal blood).2. The ;LH-free' fraction of hypophysial portal plasma from hypophysectomized and from ovariectomized rats caused a greater depletion of ovarian ascorbic acid in immature rats, pretreated with gonadotrophins, than a similar fraction of systemic plasma obtained from the same donor animals.3. The ;LH-free' fraction of hypophysial portal plasma from ovariectomized rats evoked a rise in the level of LH in the systemic plasma of ovariectomized, oestrogen and progesterone treated, rats. This fraction also caused ovulation in rabbits when infused directly into the anterior pituitary gland of these animals. The activity of the ;LH-free' fraction of systemic plasma was considerably less than that of portal plasma in either of these assay systems.4. The results of these experiments suggest the presence of a factor in the ;LH-free' fraction of hypophysial portal plasma which is capable of causing the release of luteinizing hormone from the anterior pituitary gland. The molecular weight of this factor, as assessed by its behaviour on filtration through ;Sephadex G-25', is probably less than 5000.5. The LRF activity of the ;LH-free' fraction of hypophysial portal plasma obtained from rats at various phases of the oestrous cycle was measured by the ovarian ascorbic acid depletion method. There appears to be a decrease in the level of activity at oestrus. However, a rise in LRF activity, which was expected to occur at the ;critical period' of prooestrus, was not evident. The significance of these findings is discussed.