Nulliparous Holstein heifers were examined ultrasonically once daily during an interovulatory interval (ovulation = Day 0). Follicles with a diameter ≥4 mm were sequentially identified. Heifers were randomized into four groups (n = 4 heifers per group): untreated control heifers and those treated on Days 0 to 3, Days 3 to 6, or Days 6 to 11. Heifers designated for treatment were given an intravenous injection, twice daily, of a proteinaceous fraction of follicular fluid (PFFF; 16 ml) prepared by extracting bovine follicular fluid with activated charcoal. Mean cessation of growth of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 5.5) than in heifers treated on Days 0 to 3 (Day 1.5) or Days 3 to 6 (Day 3.5). Mean onset of regression of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 12.0) than in heifers treated on Days 0 to 3 (Day 5.0) or Days 3 to 6 (Day 7.5). Mean cessation of growth of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 3.0) than in heifers treated on Days 0 to 3 (Day 1.2). Mean onset of regression of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 7.0) than in heifers treated on Days 0 to 3 (Day 4.8). In heifers treated on Days 6 to 11, cessation of growth and onset of regression of the dominant follicle (means, Days 5.2 and 12.0, respectively) were not significantly different from those of the controls. The hypothesis that PFFF treatment on Days 0 to 3 would cause suppression of all follicles of Wave 1 was supported. The hypothesis that PFFF treatment on Days 3 to 6 would not alter growth of the dominant follicle of Wave 1 was not supported. The mean day of detection of the dominant follicle of Wave 2 was different (P<0.005) in control heifers (Day 8.5) than in heifers treated on Day 0 to 3 (Day 5.5) or Days 6 to 11 (Day 14.2). The mean length of the interovulatory interval was shorter (P<0.05) in control heifers (20.5 d) than in heifers treated on Days 6 to 11 (23.2 d). The hypothesis that PFFF treatment on Days 6 to 11 would delay the emergence of Wave 2 was supported. The proportion of heifers with 2-wave interovulatory intervals was 3 4 for control heifers and 0 4 , 1 4 , and 4 4 for heifers treated on Days 0 to 3, Days 3 to 6, and Days 6 to 11, respectively ( 3 4 vs 0 4 , P<0.05); the remaining heifers had 3-wave interovulatory intervals. On average, in PFFF-treated heifers, follicles stopped growing 1 d after treatment was started, and Wave 2 was detected 3 d after treatment was stopped.