The aim of this study was to test the effect of oestradiol-17β (E2) on the motility, viability and the acrosomal status of bull sperm in vitro. Pooled semen from Holstein bulls were incubated in the presence of 2, 4, and 8μg E2/mL for 24h. Semen was also incubated in media without E2. During the incubation, the number of motile, viable sperm and the number of sperm possessing lost/damaged acrosomes, cytoplasmic droplets and coiled tails were counted at 0, 4, 18 and 24h of incubation. Addition of 2μg E2/mL at 18h of incubation increased the total motility over the control. The number of forward progressing sperm was increased by the supplementation of 2 and 8μg E2/mL over the control group at the 4h incubation. Lower doses of E2 (2 and 4μg/mL) did not affect viability of sperm, but a high dose of E2 (8μg/mL) caused reductions in viability at 4 and 24h of incubation. The number of sperm cells with lost acrosomes was significantly high in control group at 24h of incubation. The number of sperm cells possessing proximal and distal cytoplasmic droplets and the number of sperm cells bearing coiled tails were not altered by any of the treatments. A small dose of E2 (2μg/mL) had a beneficial effect on the motility and acrosome integrity of bull sperm in vitro. Higher dose of E2 (8μg/mL) had a detrimental effect on viability. © South African Society for Animal Science.