Two studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest dominant follicle growth resulting in emergence of a new follicular wave at a predictable interval post-treatment. In a first experiment, postpubertal beef heifers were assigned randomly to four treatment groups and given phosphate-buffered saline (controls; n=10), or letrozole at a dose of 500 (n=9), 250 (n=10), or 125 (n=10) µg/kg intravenously 4 days after follicular ablation (~2.5 days after wave emergence). In a second study, postpubertal beef heifers were assigned randomly to four treatment groups. One group received no treatment (control; n=17) and the other groups (n=9-10) were given 85 µg/kg of letrozole per day (250 µg/kg total dose), from Days 1 to 3, Days 3 to 5, or Days 5 to 7 (Day 0 = pre-treatment ovulation,) corresponding to the periods before, during and after selection of the dominant follicle, respectively. Follicular dynamics were monitored ultrasonically and blood samples were collected for endocrine assays. Follicle diameter profiles and plasma LH, FSH, and estradiol concentrations were analyzed. Additionally, during the second trial, CL diameter profiles and plasma progesterone concentrations were measured. In both studies, the diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P<0.05) and the intervals to new wave emergence and onset of regression of the extant dominant follicle were longer (P<0.05) in heifers treated with letrozole than in controls, regardless of the dose (high, medium, or low; single vs multiple) and the stage of the follicle wave in which treatments were initiated. Furthermore, during the second experiment, the mean CL diameter was larger in letrozole-treated heifers, although there were no differences in plasma progesterone iii concentrations between treated and control animals. The effects on dominant follicle and CL diameter profiles appeared to be the result of the significantly increased plasma LH concentrations observed in letrozole-treated animals during both treatment approaches (single vs multiple dose). Incomplete and inconsistent inhibition of estradiol production and the lack of a surge on FSH observed in both experiments may be a result of insufficient circulating levels of letrozole during the treatment period. In summary, a single or multiple dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release.Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a non-steroidal method for controlling ovarian function in cattle, but further studies are warranted to clarify dosage and timing of treatment to predictably affect follicular wa...