This study was undertaken to investigate the effects of storage of stallion semen in a defined milk protein extender at 5 and 15 degrees C under either anaerobic or aerobic conditions, with or without addition of the antibiotic gentamicin. Semen samples were collected from eight fertile stallions and stored for 96 h (day 0–4) and assessed daily for motility, velocity and membrane integrity (viability) using a CASA system. Samples for bacteriology assessment were taken on day 2 of storage. No significant (p > 0.05) differences in motility, velocity or viability were observed between treatments on days 0–2. On days 3 and 4, semen stored without gentamicin at 5 degrees C had a significantly (p <0.05) better semen quality compared with storage at 15 degrees C without gentamicin, irrespective of oxygen exposure. On days 3 and 4, motility and velocity were greater in samples stored at 15 degrees C with gentamicin, compared with the corresponding treatments without antibiotic (p <0.05). This effect was also evident for viability on day 4. The decline in semen quality observed at 15 degrees C most likely resulted from the effect of bacterial growth. Bacterial growth was the greatest in samples stored at 15 degrees C without gentamicin, under both anaerobic and aerobic conditions (p <0.05). Bacterial growth was inhibited by adding of gentamicin at 15 degrees C, which accordingly reduced the decline in semen quality. Addition of antibiotic to samples stored at 5 degrees C had no significant effect on any parameter analysed. In conclusion, storage at 15 degrees C can be achieved by using an extender containing the antibiotic gentamicin. Storage at 5 degrees C tended to maintain better semen quality irrespective of oxygen exposure, and did not necessitate an antibiotic treatment.