Traditional treatments of two prostaglandin F2 alpha (PGF2alpha) doses at 10-day intervals or more did not result in acceptable pregnancy rates in timed artificial insemination (TAI) programmes in ewes. An explanation might be the undefined time-period of the onset of oestrus and ovulation after the treatment. Recently a consistent interval to oestrus and ovulation was obtained by giving PGF2alpha at day 3 post-ovulation, i.e. when the largest follicle of the first follicular wave of the cycle was still growing. This can be achieved when a second dose of PGF2alpha is given 7 days after a first dose. In this work, we evaluated the synchronization of oestrus and determined which of three different moments of TAI was the most successful using a PGF2alpha (PG-7d) protocol in a large flock. A total of 436 nulliparous and multiparous ewes were treated with two doses of a PGF2alpha analogue (delprostenate 160 microg, i.m.) separated by 7 days. Onset of oestrus was recorded twice a day and a single cervical TAI with fresh undiluted semen was performed either at 42 h (n = 152), 48 h (n = 120), or 54 h (n = 164), after the second PGF2alpha dose without taking into account the oestrous response. Pregnancy rate was determined by transrectal ultrasonography 30 days after insemination. Onset of oestrus was detected in 308 of 328 and 89 of 108 multiparous and nulliparous ewes, respectively (p < 0.001), within 72 h after treatment. The distribution of the onset of oestrus did not differ between multiparous and nulliparous ewes and the highest proportion of ewes in oestrus was detected between 25 to 48 h (313/397) from the second PGF2alpha dose. The pregnancy rate in ewes inseminated at 42 h tended to be higher than those inseminated at 48 h (p = 0.09) and was higher than those inseminated at 54 h (p < 0.05) (56/152, 31/120, 37/164; respectively). Therefore, the use of the PG-7d protocol resulted in a very high synchronization of oestrus with the highest concentration (around 80%) between 25 to 48 h from the end of treatment. The best pregnancy rate (37%) was obtained after a single cervical TAI with fresh semen at 42 h.