The objective of this study was to clarify the effects of a second protocol of ovulation synchronisation starting on day 26 after timed artificial insemination on ovarian profile and days open in dairy cows diagnosed as nonpregnant. Ninety-four Holstein-Friesian cows received intramuscular injections of a GnRH analogue (GnRH), 100 microg fertirelin, on day 0 and a prostaglandin F(2alpha) analogue (PG), 5 mg etyprostontromethamine, on day 7. GnRH was again administered 48 h after the PG injection, and timed artificial insemination was performed 16 to 20 h later (Ovsynch/TAI). Twenty-six of the 94 cows returned to oestrus within 26 days after TAI and were inseminated. Of the other 68 cows, 44 were not pregnant and were randomly allocated to undergo another Ovsynch/TAI protocol (Resynch group; n=23) or AI only after detection of oestrus (Control group; n=21). The ovarian and hormonal profiles were compared between the first and second Ovsynch protocol periods in the Resynch group. The diameter of the dominant follicle and plasma oestradiol-17 beta concentration at the second GnRH injection were significantly greater than those at PG injection during the second Ovsynch period. Ovulation was synchronised in all of the animals in the second Ovsynch period. The AI submission rates, mean AI intervals and pregnancy rates of the Resynch and Control groups were 100% and 57.1%, 36.0 +/- 0.0 and 43.2 +/- 10.9 and 30.4% and 14.3%, respectively. The mean AI interval was 7 days shorter and the pregnancy rate was higher in the Resynch group than in the Control group, although no significant differences were found due to the small number of the animals. In conclusion, the Resynch protocol initiated on day 26 after TAI in the first protocol has the potential to reduce days open and increase the pregnancy rate in dairy cows.