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The objective of this study was to examine the influence of 17β-estradiol (E2) on distribution of LH and FSH isoforms during the follicular phase of the bovine estrous cycle prior to the preovulatory surges of LH and FSH. On Day 16 of the estrous cycle (Day 0 = estrus), intact controls (CONT; n = 4) were treated with prostaglandin F2α (PGF2α) to induce luteal regression and initiation of the follicular phase. Other cows were also treated with PGF2α and either ovariectomized (OVX; n = 5) or ovariectomized and given E2 implants (OVXE; n = 6) to mimic the pattern of increasing E2 concentrations during the follicular phase of the estrous cycle. Pituitaries were collected 40 h after treatment with PGF2α, or ovariectomy (0 h). Aliquots of pituitary extracts were chromatofocused on pH 10.5-4.0 gradients. The LH resolved into thirteen isoforms (designated A-L and S, beginning with the most basic form) while FSH resolved into nine isoforms (designated I-IX, beginning with the most basic form). The percentage of LH as isoform F (elution pH = 9.32 + 0.01) was greater (p < 0.05) in the OVX group (48.5%) than in the OVXE group (45.0% ). LH isoforms I (elution pH = 6.98 ± 0.01) and J (elution pH = 6.48 ± 0.01) were more abundant (p < 0.05) in cows from the OVXE (2.3 and 5.8%, respectively) than the OVX group (1.4 and 3.7%, respectively). Distribution of LH isoforms in cows from the three groups did not differ (p > 0.10). Distribution of FSH isoforms were similar (p > 0.05) among all groups. In summary, removal of the ovary (OVX) resulted in a slight increase in percentage of the basic LH isoform F, while removal of the ovary and administration of E2 (OVXE) in a pattern that mimicked increasing concentrations of E2 during the follicular phase of the estrous cycle resulted in a slight increase in the percentage of acidic LH isoforms (I and J). There was no influence of ovariectomy or treatment with E2 on distribution of FSH isoforms in the pituitary. Thus, gonadotropin heterogeneity does not appear to change significantly during the follicular phase of the bovine estrous cycle.