Flow sorting cytometry has been shown to repeatedly produce viable sexed sperm at a level necessary for commercialization. Previous reports have shown that pregnancies rates were lower using sexed semen than commercial nonsexed semen. Sperm concentration, acrosome activation, and time to ovulation are some factors explaining the low conception. The objective of the present study was to compare pregnancy rates using sexed semen (3 × 10⁶ sperm) deposited in different locations of the bovine uterus (body v. horn) and inseminated (AI) at 2 different times (52 h v. 58 h after progesterone intravaginal device removal). Holstein heifers, between 15 to 18 mo of age and a body condition score 3.05 ± 0.22 were used. All heifers were evaluated by transrectal ultrasonography to determine ovarian structures (284/357, 79.5% had a CL). On Day 0, all heifers received an intravaginal progesterone (P4) device (1.9 g of P4, CIDR, Pfizer, Buenos Aires, Argentina) plus 70 μg of D-cloprostenol (Bioprost, Biotay, Argentina) i.m. and 2 mg of estradiol benzoate (EB, Syntex SA, Buenos Aires, Argentina). On Day 8, CIDR devices were removed and heifers received 150 μg of D-cloprostenol i.m. at the same time. On Day 9, all heifers received 1 mg of EB i.m. and were randomly allocated in 4 different groups (2 × 2 factorial): group Body-52 h, AI in the body of the uterus at 52 h (n = 98); group Body-58 h: AI in the body of the uterus at 58 h (n = 104); group Horn-52 h: AI in the ipsilateral horn of the ovulatory follicle (detected by ultrasonography) at 52 h (n = 90) and group Horn-58 h: AI in the ipsilateral horn to the ovulatory follicle at 58 h (n = 65). All heifers were inseminated with only one dose of sexed semen. At time of AI an ultrasound examination was done to determine the size and the location of the preovulatory follicle. AI was done by a single inseminator using a traditional AI gun, and an embryo transfer gun (ET sheath, SBS Cryotec SA, Argentina) was used for deep AI. Pregnancy diagnosis was evaluated by transrectal ultrasonography using a 7.5-MHz transducer (Mindray 6600) 30 days after AI. Data were analyzed by logistic regression. Sixty percent of the preovulatory follicles were on the right ovary, and there were no differences between the sizes of the preovulatory follicle among groups (overall mean ± SEM: 15.5 ± 1.0 mm). Although pregnancy rates did not differ between horn (64/155, 41.2%) and body (70/202, 34.3%; P < 0.1) inseminations and between 52 h (67/188, 36.7% and 58 h (67/169, 39.6%), pregnancy rates were higher (P < 0.05) in heifers inseminated in the horn at 58 h (32/65, 49.2%) than those inseminated in the body at 58 h (35/104, 33.6%) and tended (P < 0.09) to be higher than heifers inseminated in the horn at 52 h (32/90, 35.5%) and in the body at 52 h (35/98, 35.7%). Fixed-time artificial insemination using ultrasonography and deep insemination could contribute to enhanced pregnancy rates using sexed semen in Holstein heifers.