The objectives of this trial were to determine the effects of 0.5 mg estradiol benzoate administered intramuscularly 24 h after the removal of progesterone-containing intravaginal devices on the occurrence and timing of estrus, synchronized pregnancy rate and synchronized conception rate in dairy heifers. A clinical trial was conducted involving 750 dairy heifers in 13 herds. Within each herd heifers were randomly allocated to 1 of 2 estrus synchronization treatments. All heifers received a CIDR-B progesterone-containing intravaginal device containing a 10 mg estradiol benzoate capsule for 12 d. Twenty-four hours after CIDR-B removal one group received 0.5 mg, im estradiol benzoate while the other group received an intramuscular injection of a placebo. Estrus detection was performed at 48 and 72 h after intravaginal device removal, and heifers detected in estrus at those times were inseminated. Administration of estradiol benzoate 24 h after removal of CIDR-B devices significantly increased the number of heifers exhibiting estrus within the observation period (96.1 vs 90.5%; P < 0.01). It also altered the onset of estrus so that significantly more heifers were in estrus (86.6 vs 72.3%; P < 0.01) and conceived (47.1 vs 37.5%; P < 0.05) by 48 h after CIDR-B device removal. The synchronized conception rate was unaffected by treatment. The distribution of estrus was such that fixed-time insemination after estrus synchronization with this treatment program may be recommended.