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This study aimed to determine the effect of antral follicle count (AFC), and pubertal status on the fertility of beef heifers. In this study, 230 Nelore heifers, 20±2 months of age, were subjected to an estradiol progesterone-based timed artificial insemination (TAI) program. On Day 0 of the TAI protocol, the heifers were examined by transrectal ultrasound to record videos of the ovaries. Later, in the darkroom of the laboratory of images, the videos were analyzed for AFC (≥ 3 mm) of each ovary. Females who failed the first TAI were resynchronized with the same hormonal protocol. The pregnancy status was evaluated by ultrasonography 30 days after each FTAI. The general mean of the AFC was 22.0 follicles. Thus, the heifers were divided into 2 groups according to AFC: Low AFC (˂ 22 follicles, n = 114), and High AFC (≥ 22 follicles, n = 116). No differences (P > 0.05) in the pregnancy per AI (P/AI) were observed between the Low and High AFC groups, and between pubertal and prepubertal categories. The P/AI was not different between heifers that displayed or did not estrus (P = 0.2). However, considering the estrus response of each AFC group, High AFC heifers that displayed estrus had greater P/AI (P = 0.01) than High AFC heifers that did not display estrus. In summary, AFC and pubertal status did not affect the fertility of Nelore heifers. In contrast, the P/AI of heifers that did not display estrus was lower than heifers observed in estrus only in the High AFC group.