Meiotic maturation of the oocyte is a timed sequence of events induced by the ovulatory LH surge. In vitro maturation of oocytes is known to alter the meiotic time course. This study documented the timing of meiosis in oocytes grown in vitro for 12 days, from the preantral follicle stage onward, and the influence of an oil overlay. In the oil-free culture, the stability of the metaphase II spindle was further explored to determine the postovulatory aging events. After the maturation stimulus, in vitro-grown oocytes were collected at 2-h intervals spanning the period of meiosis (0-18 h) and at 3-h intervals during early postovulatory aging (18-27 h). Stage of maturation was assessed both morphologically and by detailed spindle analysis and chromosome alignment. Results revealed that oil overlay did not impair the competence of cultured oocytes to proceed to meiosis II, but delayed meiosis I progression. Oil overlay during culture causes a different hormonal exposure of the follicles by a differential segregation into the oil overlay. The use of a progesterone receptor antagonist, however, did not induce a delay in meiotic progression. Aging effects in oil-free cultured follicles were detected 5 h after the establishment of the metaphase II spindle, comparable to their in vivo grown counterparts. The predominant effect of aging was an interphase-like appearance of the cytoskeleton. So an optimal time window for fertilization after in vitro follicular growth was determined to be 16-21 h after maturation induction.