Peripubertal beef heifers (n = 57) and postpartum multiparous cows (n = 52) were used to determine the optimal dose of estradiol benzoate (EB) to induce and synchronize estrus after treatment with intravaginal progesterone inserts (IVP4, EAZI-BREED CIDR). All females received an IVP4 for 7 d (d 0 = insertion day) with a 25-mg injection of PGF2alpha (Lutalyse) on d 6. At 24 to 30 h after IVP4 removal, females were randomly assigned to be injected subcutaneously with EB at the following doses: heifers 0, .2, .38, or .75 mg and cows 0, .25, .5, or 1 mg. Furthermore, seven heifers and seven cows from each dose group were bled every 4 h for 76 h starting at EB injection. Serum was collected and assayed for LH and estradiol-17beta (E2). Observations for signs of estrus were made twice daily for 21 d after removal of IVP4, and females were artificially inseminated 8 to 20 h after detection of estrus. The percentage of females showing estrous behavior was increased by EB (P < .04); the greatest response was at .38 mg in heifers (86%) and 1 mg in cows (100%). Dose x time interaction affected (P < .01) E2 concentrations in heifers and cows; the animals that received the higher doses of EB had greater E2 concentrations in a shorter time than those that received the smaller doses. The percentage of cows and heifers with an acute preovulatory LH release (peak LH) was affected by dose, with a linear (P < .01) and a quadratic (P < .01) response. Highest concentrations of LH during peak LH were affected by dose with a linear (P < .01) response in heifers and linear (P < .01) and quadratic (P < .08) responses in cows. Heifers receiving .38 mg and cows receiving .5 and 1 mg of EB had the highest peak LH. Time to LH peak had a linear (P < .03) response in heifers and had linear (P < .04) and quadratic (P < .05) responses in cows. Pregnancy rate was affected (P < .02) in heifers by whether or not they were anestrous before IVP4 treatment (those with estrous cycles = 52% vs those that were anestrous = 22%) and in cows by dose of EB (P < .01; 8, 23, 21, and 67% for 0, .25, .5, and 1 mg, respectively). In conclusion, in females treated with IVP4 and PGF2alpha to induce and synchronize estrus, an injection of EB increased concentrations of E2 and LH and increased number of animals showing estrus. Also, EB increased pregnancy rates in cows. Optimal responses were at .38 mg EB for heifers and at 1 mg EB for cows.