There are reports in the literature indicating that use of eCG improves pregnancy rates in Bos indicus anestrous cows treated with a progesterone-based fixed-time AI (FTAI) protocol. More recently, replacement of eCG by FSH in FTAI protocols was reported to have beneficial effects (Bos indicus) or no effect (Bos taurus) on pregnancy rates. In the present experiment the effects of eCG and FSH on pregnancy rates were compared in an FTAI protocol. Primiparous lactating Nellore cows (40 to 80 days postpartum, n = 421) with a body condition score of 2.5 to 3.0 (on a 1 to 5 scale) were randomly allocated in 3 groups: Control (CTR), eCG, and FSH. In the control group, all animals received a progesterone (P₄)-releasing intravaginal device (1.55 g, PRID^®, Ceva Sante Animale S.A., Libourne, France) and 2.5 mg of estradiol benzoate (EB, i.m. Estrogin^®, Farmavet, São Paulo, Brazil), on Day 0 (D0). Eight days later (D8), at the time of the intravaginal device withdrawal, PGF_2α (150 μg, D-cloprostenol, i.m. Prolise^®, ARSA S.L.R., Buenos Aires, Argentina) was administered. Twenty-four hours after PRID removal, cows were treated with EB (1.0 mg, i.m.), and FTAI was done 30 to 36 h later. In the eCG and FSH groups, the cows were treated with 20 mg of FSH (Folltropin-V^®, i.m. Bioniche, Belleville, Canada) or400IU of eCG (Novormon^®, i.m. Syntex, Buenos Aires, Argentina), respectively, at the time of PGF_2α administration. Ovarian ultrasonography (Aloka SSD 500, 7.5-MHz probe, Aloka, Tokyo, Japan) was performed 10 days prior to and at the beginning of FTAI protocol to select the animals in postpartum anestrous (absence of CL in both examinations). The pregnancy diagnosis was performed by ultrasonography 30 days after FTAI. Data were analyzed by logistic regression (SAS Institute, Cary, NC, USA). Cows treated with eCG had a higher pregnancy rate (41.5%, 95/229; P < 0.05) than those treated with FSH (22.7%, 22/97) or in the control group (26.3%, 25/95). Pregnancy rates of animals treated with FSH did not differ (P > 0.05) from those in the control group. These results indicated that, in lactating primiparous anestrous Nellore cows, the use of eCG in a FTAI protocol improves the pregnancy rate, whereas FSH has no beneficial effect.