The appearance and production of an early pregnancy factor (EPF) has been studied in sheep and cattle. This factor can be detected in serum and tissues of pregnant animals by its synergistic action with antilymphocyte serum in reducing the number of rosettes formed in a rosette inhibition test. The range of the rosette inhibition titre for serum from non-pregnant animals was 4-10. Values higher than these were considered to indicate the presence of EPF. In one ewe EPF was detected 29.5 h after the onset of oestrus at which time a zygote was recovered. The transfer of sheep zygotes into unmated non-pregnant ewes, and the infusion of extracts of mouse zygotes into the oviducts ipsilateral to the side of ovulation in ewes caused an increase in the titre of serum from the recipient animals. Infusion of extracts of unfertilized mouse ova did not change the titre. Removal of zygotes or embryos resulted in a reduction in the titre within 48 h to non-pregnant values. Extracts of tissues analysed for the presence of EPF gave high titres when taken from early pregnant ewes whereas tissues from non-pregnant animals gave low values. Removal of the ovary containing the corpus luteum from sheep 4-5 days after mating and in which pregnancy was maintained by exogenous progesterone resulted in a decrease in titre during the following 2-4 days. Removal of the contralateral ovary or the induction of luteolysis with a prostaglandin F-2a analogue did not result in a decrease. In cattle, pregnancy diagnosis by the rosette inhibition test 4 days after an artificial breeding programme gave good agreement with conventional pregnancy testing methods, e.g. palpation per rectum and plasma progesterone assays. From these experiments we suggest that a substance 'zygotin' is released by the fertilized egg and stimulates the production of EPF, most probably from the ovary containing the corpus luteum. Zygotin may act directly on the ovary or via an intermediary substance released by the oviduct. The role of EPF is unknown. It does not appear to be necessary for the maintenance of pregnancy because ovariectomized sheep with no demonstrable EPF remain pregnant when given exogenous progesterone. Routine pregnancy testing in sheep and cattle is not practicable with the present rosette inhibition test.