Low molecular weight insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, are believed to inhibit the actions of insulin-like growth factors (IGFs). We showed previously that ovarian follicular dominance in cattle is associated with the presence of a protease that degrades IGFBP-4. To test the hypothesis that specific IGFBP-4 proteolysis is associated with selection of the dominant follicle, we induced codominant follicles (co-DFs) during the first follicular wave of the estrous cycle. The ovaries of Holstein heifers were examined twice daily by ultrasonography; when the largest follicle reached 6 mm in diameter, saline (control, n = 5) or 2 mg of recombinant bovine (rb) FSH (FSH, n = 5) was injected i.m. every 12 h for 48 h. Follicular fluid was collected by aspiration from the two largest follicles/heifer 12 h after the last injection. IGFBPs in follicular fluid were quantified by Western ligand blotting/phosphorimaging. IGFBP-4 protease activity was measured by incubating follicular fluid with recombinant human (rh) IGFBP-4 substrate, followed by ligand blotting/phosphorimaging to quantify the percent of substrate loss and Western immunoblotting to detect specific proteolytic fragments. Co-DFs of FSH heifers did not differ (P > 0.05) from the single dominant follicle of controls in size, or in concentration of progesterone or level of IGFBP-4 in follicular fluid. In contrast, the largest subordinate follicle of control heifers was smaller, with lower progesterone and higher IGFBP-4 in the follicular fluid (P < 0.05). Concentrations of estradiol in follicular fluid were high in dominant follicles, intermediate in co-DFs, and low in subordinate follicles (P < 0.05). IGFBP-4 protease activity in co-DFs was similar (P > 0.05) to that of dominant follicles, but fourfold higher (P < 0.05) than that of subordinate follicles. The results strongly suggest that an FSH-dependent IGFBP-4 protease is associated with selection of the dominant follicle in cattle.