Fixed time insemination requires both the functional life span of the corpus luteum to be regulated and follicle wave status to be synchronised in all animals, irrespective of the stage of oestrous cycle or follicle wave. The objective of this experiment was to compare the oestrous response in heifers given either GnRH or ODB at the start of a progesterone treatment. Treatments were initiated at emergence or dominance of the first follicle wave (days 2 and 5 of the oestrous cycle, respectively) or at emergence or dominance of the second follicle wave (days 10 and 13, respectively). The second objective was to compare the oestrous response in heifers given a luteolytic agent one day before the end of a progesterone treatment of 8 days duration with ODB at the start of treatment compared with a progesterone treatment of 10 days duration with ODB at the start of treatment and without using a luteolytic agent. Charolais cross-bred beef heifers (n=134) were randomly assigned to one of 3 treatments; 0.75 mg ODB given at insertion of a progesterone releasing intra-vaginal device (PRID) treatment of 10 days duration (10dE2), 0.75 mg ODB at insertion of a PPJD treatment of 8 days duration with 15mg luprostiol given one day before PRID removal (8dE2) or 250 fig GnRH at insertion of a PRID treatment of 8 days duration with 15 mg luprostiol given one day before PRID removal (8dGnRH), initiated on days 2, 5, 10 or 13 of the oestrous cycle. Oestrous detection was carried out six times daily from 24 hours after PRID removal. The proportion of heifers detected in oestrus was higher (P<0.05) for heifers in the 8dE2 treatment (40/40) compared with heifers in the GnRH treatment (38/42) and tended to be higher (P=0.08) compared with heifers in the 10dE2 treatment (38/41). The day of cycle on which treatment was initiated did not affect (P>0.05) the proportion of heifers that were detected in oestrus. The median onset of oestrus was earlier (P<0.05) for heifers in the 10dE2 treatment group (median 41 h, range 92 h) compared with either the 8dE2 (median 49 h, range 64 h) or 8dGnRH heifers (median 49 h, range 92 h). The variation in onset of oestrus tended to be decreased (P=0.075) in 8dE2 heifers compared with the 10dE2 heifers but was not different (P>0.05) to heifers in the 8dGnRH treatment group. In conclusion, 0.75 mg ODB given at insertion of a progesterone treatment of 8 days duration with 15 mg luprostiol given one day before progesterone removal increased the proportion of heifers detected in oestrus compared with heifers given 250 μg GnRH at the start of treatment. There was no difference in the efficacy of ODB or GnRH given at insertion of a progesterone treatment of 8 days duration to synchronise oestrus in beef heifers. The use of a luteolytic agent given one day before removal of a progesterone treatment of 8 days duration was more effective to synchronise oestrus than a progesterone treatment of 10 days duration without the use of a luteolytic agent.