Deviation during a follicular wave in mares begins when the largest follicle (F1) reaches a mean diameter of 22.5 mm and is characterized by continued growth of F1 to become the dominant follicle and regression of F2 to become the largest subordinate follicle. In the present study, F1 was ablated at the expected beginning of deviation (Hour 0) to provide a reference point for characterizing the intrafollicular changes preceding experimental deviation between F2 and F3. Diameters and concentrations of follicular fluid factors in F2 and F3 were determined in F1-ablated mares at Hours 0, 12, 24, 48, or 72 (n = 8 mares/group). Circulating FSH concentrations were greater (P < 0.05) in the Hour 72 ablation group than in controls 12 h after ablation and then progressively decreased. The diameters of F2 and F3 increased (P < 0.05) during Hours 0 to 24. Thereafter, F2 continued to increase but F3 did not, indicating that experimental deviation began at Hour 24. The diameter of F2 and circulating FSH concentration at Hour 24 were similar (P > 0.1) to the diameter of F1 and FSH concentration at Hour 0, respectively. A differential change between F2 and F3 was not detected in follicular fluid concentrations of estradiol, inhibin-A, and activin-A by the beginning of experimental deviation. However, estradiol was higher in F2 at Hours 0 and 12 and inhibin-A was higher in F2 throughout the experiment, and both factors could have been involved in experimental deviation. Free insulin-like growth factor-1 (IGF-1) increased (P < 0.05) in F2 beginning at Hour 12 and was higher (P < 0.05) in F2 than in F3 by the beginning of experimental deviation. Temporally, this result indicated that intrafollicular IGF-1 was involved in conversion of F2 from a destined subordinate follicle to a dominant follicle.