This study evaluated the relationship between proestrus length and follicular size, estrous behavior, and pregnancy rate in Bos taurus beef heifers subjected to ixedetime artificial insemination (FTAI). A total of 911 heifers received a synchronization treatment protocol for FTAI (JeSynch) consisting of an intravaginal progesterone device for 6 d, estradiol benzoate at the time of device insertion cloprostenol sodium and eCG at device removal and GnRH at the time of FTAI. The presence or absence of a corpus luteum (CL) was determined by ultrasonography at device insertion and all heifers were tail painted at device removal for estrus detection at the time of FTAI. For the establishment of different periods of proestrus length (i.e., interval from device removal to FTAI), GnRH was administered i.m. at 48 h (n ¼ 308), 60 h (n ¼ 290) or 72 h (n ¼ 313) after device removal. The diameter of the largest follicle at the time of GnRH administrationwas determined by ultrasonography, expression of estrous was determined by percentage of tail paint removal, and FTAI was performed at the time of GnRH administration in all heifers. The diameter of the largest follicle was greater when GnRH/FTAI was performed at 72 or 60 h (12.9 ± 0.2 mm and 12.8 ± 0.1 mm, respectively) than at 48 h (12.2 ± 0.1 mm, P