The improvement of efficiency of methods to mature equine oocytes in vitro is necessary for the application of biotechnologies in equine reproduction. Two maturation media, TCM-199 and follicular fluid (FF), were compared to mature equine cumulus-oocyte complexes (COCs) in vitro. Nuclear maturation and mitochondria distribution, and the ability to develop into embryos after transfer to inseminated mares were used as end points. In compact COCs, the percentage of oocytes that resumed meiosis (Metaphase I and II) was significantly higher in FF (77.3%) than in TCM-199 (46.8%). Compact COCs matured in FF reached a significantly (P < 0.05) higher percentage of MetaphaseII oocytes (31.8%) than those matured in TCM-199 (12.8%). Stages of nuclear maturation were associated with different patterns of mitochondrial distribution; 52.6% (40/76) of the oocytes found at germinal vesicle had dense clusters of mitochondria in the cortical area, whereas most oocytes at Metaphase I (63.6%, 42/66) showed either non-polarized or polarized patterns of mitochondria. Similarly, 76.7% (33/43) of Metaphase-II oocytes also had these patterns. After intrafollicular or intra-oviductal transfer to inseminated mares, few oocytes matured in FF (4.4%, 3/68) and developed to embryos in vivo compared to oocytes matured in TCM199 (0/58). In conclusion, pure equine FF was suitable as an in vitro maturation medium, inducing higher rates of nuclear maturation than a semi-defined medium. In addition, distribution of mitochondria changed during maturation from dense clusters of mitochondria in the cortical region in germinal-vesicle oocytes to a more dispersed pattern in Metaphase-I and -II oocytes.