We analysed the results of insemination (AI) with 2.2 million X-chromosome-bearing frozen–thawed sperm in 2283 Estonian Holstein (EHF) heifers on 7 dairy herds. The heifers of 11 to 18 months of age were inseminated with sexed sperm or unsexed control semen doses (15 × 106 sperm) from 10 different bulls either 1) at fixed time following synchronization of oestrus by 2 injections of PGF2α, 2) at visually detected spontaneous oestrus or 3) at oestrus displayed after a single injection of PGF2α. At AI, the presence and intensity of estrous signs (vulvar edema, hyperemia, discharge of mucus and an ease to pass through the cervix by catheter) were recorded. Pregnancy status of heifers was diagnosed by rectal palpation of the uterus 45–60 days after AI. Statistical analyses were performed using the SAS package (1999; SAS Institute Inc., Cary, NC, USA). The pregnancy rate of heifers after fixed-time AI at synchronized oestrus was 42.7%, which is about 80% of control unsexed semen doses (53.0%, P < 0.05). The pregnancy rate after intracornual deposition of sexed sperm (44.1%) did not differ (P > 0.05) from conventional insemination into the uterine body (41.9%). The pregnancy rates after AI of heifers with sexed sperm at spontaneous oestrus (55.9%) or oestrus displayed following a single PGF2α treatment (50.8%) did not differ between each other, but were higher than at fixed-time AI (42.7%; P < 0.05). The pregnancy rates after AI with sexed sperm at spontaneous oestrus and induced oestrus were about 90% and 85% of that of unsexed semen doses, respectively (P < 0.05). The pregnancy rates varied among the farms and among the bulls. For some farms and bulls the pregnancy rates with sexed sperm doses were similar to the pregnancy rates with unsexed regular semen doses. The pregnancy rates did not differ between heifers housed in tie-stalls or free-stalls. Pooled across sperm deposition sites, heifers that showed strong estrous signs at fixed-time AI with sexed sperm had 2.7 times higher pregnancy rate than heifers with weak signs of oestrus. The difference in pregnancy rates was 1.2 times higher in heifers with strong estrous signs when unsexed semen was used at fixed-time AI (P < 0.05) and when the heifers were inseminated with sexed or unsexed sperm doses at visually detected spontaneous or induced oestrus. The mean age and body weight did not differ between the heifers that conceived and those that failed irrespective of AI treatment. In conclusion, insemination of heifers with sexed sperm at spontaneous oestrus or oestrus induced by PGF2α treatment resulted in higher pregnancy rates than insemination at fixed time after oestrus synchronization. Intracornual deposition of semen did not improve the pregnancy rate. However, good reproductive status of a herd along with the selection of heifers according to the intensity of oestrus expression improves the efficiency of using sexed sperm for the insemination of dairy heifers. The study was supported by the ESF grant 7814.