The aim of this research was to study the effect of incubation time on capacitation and acrosome reaction of in vitro ovine spermatozoa. Twelve ejaculates from two Garut sheep were collected using artificial vagina and then evaluated macro and microscopically. After semen washing (centrifugation method), semen was diluted in Brackett and Oliphant (BO) medium and then incubated during 6 hours in CO2 incubator at 38.5 degrees C. Evaluation of capacitation and acrosome reaction was conducted on 0 hour, 0.5 hour, 1 hour, 2 hours, 4 hours and 6 hours incubation time. The result showed that incubation time had significant (P<0.05) effect on no capacitation sperm (pattern F), capacitation (pattern B) and acrosome reaction (pattern AR). Incubation time had significantly (P<0.01) affect the subjective motility, life sperm and membrane integrity.