The aim of the present study was to compare the levels of 17β-estradiol, testosterone, progesterone, inibin, and nitric oxide in follicular fluid between superovulated mares (G1) using equine pituitary extract (EPE) and nonsuperovulated mares (G2) used as control. Two estrus cycles from 24 mares (G1 = 12; G2 = 12) were monitored. In G1, the first cycle was used to determine the ovulation day (Day 0), followed by the administration of PGF2 (250 μg daily of cloprostenol; Sincrocio™, Ouro Fino, São Paulo, Brazil) on the first and second day of treatment with EPE. During the second cycle, EPE (25 mg i.m.) was administered twice daily from Day 7 to the ultrasound detection of a majority of follicles at least 35 mm in diameter. At this time, 2500 IU of hCG IV (Vetecor™, Hertape Calier, Minas Gerais, Brazil) was given. The same protocol was used for G2 as was used for G1 except that EPE administration was replaced by saline injections. For follicular aspiration, the mares were sedated with xilazine (0.5 mg kg^-1; Sedazine™, Fort Dodge, São Paulo, Brazil) and acepromazine (0.05 mg kg^-1; Acepram 1%™, Univet, Milton, Ontario Canada); hioscin bromide (Buscofin™, Agener União, São Paulo, Brazil) was also administered to reduce intestinal motility and to make ovary manipulation by way of the rectum easier. All follicles larger than 35 mm were aspirated using a Cook^® double lumen needle (12 GA) in a transvaginal aspiration probe guided by ultrasonography. The number of aspirated preovulatory follicles was 4.75 ± 2.7 and 1.0 ± 0.0 for G1 and G2, respectively. The follicular fluid collected was centrifuged at 600 × g for 10 min in order to separate the fluid from the cells. The fluid was stored at -20°C and sent to the University of California (Davis, USA) for hormonal assay. The nitric oxide dosage was determined at São Paulo State University (Botucatu, Brazil) using the luminescent reaction of Griess. The comparison between groups of hormonal levels and nitric oxide was performed using the t-test at 5% significance. The results of the present study showed that the EPE treatment (G1), under the experimental conditions, did not lead to a significant change in the mean concentrations of the evaluated hormones or of nitric oxide in the follicular fluid when compared with the levels in G2. However, differences were found among the individuals only in G1 in all hormones (P < 0.05), with exception of progesterone (P > 0.05).