In order to study the initial formation of the vitelline envelope and the appearance of vitellogenin in oocytes of rainbow trout, females were sampled monthly from 19 to 5 mo before ovulation. Immunohistochemistry revealed that the formation of the vitelline envelope starts when the oocytes reach a diameter of about 450 microns. Oocytes of this size were first found in females sampled a year before ovulation at the time when plasma levels of estradiol-17 beta increased from 0.2 to 0.6 ng/ml. An antiserum directed against vitellogenin crossreacted with small vesicles (around 2 microns) present just inside the oolemma, when the oocytes reached a diameter of 600 microns. This was interpreted as an active uptake of vitellogenin. Oocytes of this size were first found in females sampled 9 mo before ovulation at the time when estradiol-17 beta levels increased from 0.6 to 1.0 ng/ml and the gonadal somatic index was doubled. Oocytes with a diameter of 600 microns had an immunoreactive vitelline envelope with a thickness of about 3 microns. It is apparent that the initial formation of the vitelline envelope starts before the active uptake of vitellogenin and that the low previtellogenic plasma levels of estradiol-17 beta observed in females are of physiological significance.