Seminal storage, both fresh and cryopreserved, has contributed to the reproduction of wild birds in captivity, however, a method of predicting the fertilization capacity is needed. Indicators of the sperm acrosome reaction (AR) have been associated with its fertilization capacity. Therefore, the aim of this study was to evaluate the in vitro AR of fresh and cryopreserved Harris hawk sperm using Beltsville Poultry Semen Extender (BPSE) and Lake as diluents. 64 ejaculates were obtained and examined fresh and after thawing. The sperm basic evaluation for each ejaculate was made using eosin-nigrosin, while the ability of AR was assessed by co-incubation with perivitelline layer (PVL). Sperm motility of fresh semen was higher (P<0.05) in fresh semen in BPSE (37.9±1.7) than in Lake (30.9±1.7) diluent. However, the motility decreased (P<0.05) in both diluents after thawing. For fresh semen, the percentage of sperm that underwent an AR without incubation with PVL was higher (P<0.05) with BPSE (14.1±1.7) than with Lake (6.8±2.5) diluent, however, AR was similar between tow diluents (P>0.05) after thawing. The percentage of sperm that underwent an AR when incubated with PVL post thawing in Lake (45.4±2.7) was lower (P<0.05) than that of fresh semen (55.3±3.1), whereas there were no differences (P>0.05) with BPSE. It is concluded that Lake diluent was more efficient for fresh seminal storage, while BPSE diluent was more efficient for seminal cryopreservation in Harris hawk.