This study compares the effects of reduced (5%) or normal (5% CO2 in air; 20% O2) oxygen tension on the in vitro maturation of early preantral ovarian follicles isolated from 14-day-old (C57BI/6J x CBAca) F1 mice. Intact follicles (100-130 microns) are singly cultured in 20 microliters droplets alpha-MEM enriched with FCS and rFSH under mineral oil at 37 degrees C and 100% humidity. In this culture system the follicles are allowed to attach to the bottom of the petri dishes. Follicle in vitro growth, hormone secretory capacity, and in vitro ovulation were studied under the two oxygen tensions. Spontaneous oocyte release from the follicle during a 16-day culture period was observed significantly more under 5% oxygen. Antral-like cavity formation was not observed under 5% O2. The follicles in the 5% O2 cultures reaching day 16 were stripped of their granulosa cell layers, and 83% of the retrieved oocytes had already undergone spontaneous germinal-vesicle breakdown (GVBD). Under 20% O2, the GV stage was maintained until day 16 in 77% of the oocytes. Under 5% O2, intact follicle survival up to day 12 was significantly reduced as compared to the 5% CO2 in air conditioning. The hCG stimulus on day 12 induced mucification in a significantly larger proportion of follicles cultured under 20% O2 (79% vs. 47%). Germinal-vesicle breakdown (20% O2:95, 5%, O2:42%) and first polar body extrusion (20% O2:40%, 5% O2:15%) were significantly more prevalent under normal oxygen tension. A reduced secretory capacity of E2 and inhibin was demonstrated for follicles cultured under 5% O2. The histological study of serially sectioned follicles showed increased areas of centrally located granulosa cell necrosis and pyknosis in the cumulus cells. Gassing follicle cultures using 5% CO2 in air provided appropriate conditions for normal growth, enhanced whole-follicle survival, differentiation, and hormone production, and improved the yield of meiotic competent oocytes.