Oestrus synchronization following prostaglandin-induced luteolysis is variable and dependent on follicle wave status in cattle. Oestradiol benzoate (ODB) has been used following prostaglandin to reduce the interval to oestrus and ovulation, but the effect of follicle wave status at the time of ODB administration is not clear. The aim of this study was to characterize the endocrine and follicular responses following ODB after luteolysis at different stages of the follicle wave. Prostaglandin was administered at either emergence or dominance of the second follicle wave. Twenty-four hours later animals received either 0.5mg ODB in oil or a control oil injection. Follicular development was monitored daily by ultrasonography, oestrous behavior was determined and blood samples were collected. In animals treated with ODB at emergence, there was a reduction (P<0.05) in the maximum diameter of the ovulatory follicle (11.7+/-1.2 mm versus 13.1+/-0.1 mm) and in the interval from prostaglandin to oestrus (52.0+/-2.3 h versus 88.0+/-9.6h), to the LH surge (53.3+/-3.5 h versus 89.1+/-6.5 h) and to ovulation (96+/-0.0 h versus 129.6+/-9.6h), compared with controls. In animals treated with ODB at dominance, there was a reduction (P<0.05) in the interval from prostaglandin to the LH surge (54.0+/-3.1 h versus 70.9+/-4.8 h), but not in the interval from prostaglandin to oestrus (53.3+/-2.7 h versus 65.7+/-4.5 h; P=0.11), to ovulation (96.0+/-0.0 h versus 110.4+/-4.8 h; P=0.12) or the maximum diameter of the ovulatory follicle (12.7+/-0.3 mm versus 13.6+/-0.4 mm; P=0.12), compared with controls. Treatment did not affect (P>0.05) the length of the subsequent oestrous cycle or corpus luteum size. In conclusion, the use of ODB advanced, but did not alter the temporal relationships among oestrus, the LH surge and ovulation, regardless of stage of follicle development at treatment.