Three experiments were carried out to investigate whether bovine amniotic fluid (BAF), when added as a protein supplement to the maturation medium, supports the in vitro maturation of pre-ovulatory bovine oocytes. In the first experiment, the medium was supplemented with three different concentrations of the BAF (10, 20 and 50%, respectively). In the second experiment, the effects of a combination of two protein supplements BAF plus oestrus cow serum (BAF-OCS) and BAF plus bovine follicular fluid (BAF-BFF), on the in vitro oocyte maturation rates were evaluated. In the third experiment, the effects of three different protein supplements (BAF, OCS, and BFF) on the oocyte maturation percentages were compared. Maturation rates at the metaphase II stage, following supplementation with 20% BAF (77%) and 10% BAF (68%) were significantly higher (P < 0.001 and P < 0.05, respectively) than those resulting from supplementation with 50% BAF (44%). Significant differences between the 10% BAF and 20% BAF treatments were not observed. Significant differences were, however, found between the BAF-OCS treatment (57%) and the BAF-BFF treatment (50%). The results obtained from the BAF and the OCS treatments (77 and 78%, respectively) were significantly higher (P < 0.05) than when BFF (60%) was used. It is concluded that supplementation of the in vitro maturation medium of pre-ovulatory bovine oocytes with BAF provides important levels of proteins and growth factors responsible for nuclear and cytoplasmic development.