Treatment with letrozole, a non-steroidal aromatase inhibitor, has been associated with elevated mean plasma LH concentrations, a prolonged period of dominance of the extant dominant follicle, and delayed emergence of the next follicular wave in cattle. As well, a luteotrophic effect was hypothesised by the observation that CL diameter was increased in heifers given 250 μg kg–1 of letrozole divided in a 3-day regimen. The objective of the present study was to determine the effects of vehicle and route of administration of letrozole on ovarian function in sexually mature beef heifers. Ovarian function was synchronized among heifers using transvaginal ultrasound-guided follicular ablation followed by a luteolytic dose of PGF b.i.d. 4 days later. The ovaries were subsequently examined daily by transrectal ultrasonography until ovulation was detected. On Day 3 (Day 0 = ovulation), heifers were assigned randomly to 4 treatment groups and given letrozole at a dose of 1 mg kg–1 intravenously (i.v. in benzyl alcohol, n = 10) or intramuscularly (i.m. in benzyl alcohol plus canola oil 1:1 v/v, n = 10), or given a placebo (i.v. in benzyl alcohol, n = 5) or (i.m. in benzyl alcohol plus canola oil 1:1 v/v, n = 5). The ovaries were monitored daily by ultrasonography, and blood samples collected twice daily by jugular venipuncture from pre-treatment to post-treatment ovulations. Comparisons among groups were made by 1-way ANOVA for single-point measurements and by ANOVA for repeated measures for time-series data. The interovulatory interval did not differ among groups, nor did the day-to-day diameter profile of the dominant follicle of wave 1 (first follicular wave after ovulation). However, the interval between emergence of waves 1 and 2 was longer in heifers treated with letrozole i.m. (11.7 ± 0.3 days) than in controls (10 ± 0.4 and 9.5 ± 0.5 days for i.v. and i.m. controls, respectively; P < 0.05), and intermediate in heifers given letrozole i.v. (10.6 ± 0.30 days). The day-to-day diameter profile of the corpus luteum was greater (P < 0.05), and plasma progesterone concentrations tended to be greater (P < 0.06) in heifers treated i.m. with letrozole v. placebo. Plasma LH concentrations did not differ among groups, whereas plasma FSH concentrations were greater (P < 0.02) in heifers treated i.v. with letrozole v. placebo. In summary, letrozole dissolved in benzyl alcohol and given intravenously at a dose of 1 mg kg–1 on Day 3 did not alter ovarian function in cattle, but the same dose given i.m. in canola oil vehicle resulted in a longer inter-wave interval, a greater CL diameter profile, and greater plasma progesterone concentrations. We conclude that i.m. letrozole in oil is a feasible route of administration and vehicle for the development of an aromatase inhibitor-based treatment for herd synchronization in cattle. Research supported by the Natural Sciences and Engineering Research Council of Canada, Canadian Institutes for Health Research, and Bioniche Life Science Inc.