Oocytes removed from, or retained within, non-atretic and atretic follicles of different sizes were cultured for 24 h in the presence of a variety of hormones in an attempt to identify the factors affecting oocyte maturation in vitro. Resumption of meiosis was assessed morphologically; the developmental capacity of oocytes after culture was determined by transfer to the oviducts of inseminated ewes. About 70% of oocytes cultured after removal from follicles of different sizes resumed meiosis in vitro, but they did not undergo normal development after transplantation. Oocytes cultured within the follicle in hormone-free medium remained at the germinal vesicle stage. In the presence of FSH [follicle stimulating hormone] and LH [luteinizing hormone] some oocytes reached the 2nd meiotic metaphase: 19% in small (2-3 mm diameter) and 73% in larger (3-5 mm diameter) non-atretic follicles and 54% in small and 45% in larger atretic follicles. Less than 5% of oocytes cultured in follicles developed into normal blastocysts after transplantation when either no hormone or only FSH and LH were added to the culture medium. The addition of estradiol-17.beta. to medium containing FSH (2 .mu.g/ml) and LH (1 .mu.g/ml) resulted in the development to blastocysts of 26% of oocytes from small non-atretic follicles, 46% from large non-atretic follicles and 50% from atretic follicles. Blastocyst formation was greatly depressed and fragmentation rate significantly increased with concentrations of 10 .mu.g FSH/ml and 2 .mu.g LH/ml. Developmental capacity after culture was further demonstrated by the birth of lambs from 63% of blastocysts derived from oocytes matured in vitro; 52% of control blastocysts developed to lambs after transfer.