Cows that exhibit estrus within 24 h of fixed-time AI have elevated concentrations of estradiol and greater pregnancy rates compared with cows not in estrus. Our objective was to determine whether estradiol, estrus, or both had an effect on uterine pH during a fixed-time AI protocol. Beef cows were treated with the CO-Synch protocol (100 mircog of GnRH on d -9; 25 mg of PGF(2alpha) on d -2; and 100 mircog of GnRH on d 0). One-half of the cows received an injection of estradiol cypionate (ECP; 1 mg) 12 h after PGF(2alpha). Cows detected in standing estrus within 24 h of the second GnRH injection were considered to be in standing estrus. Uterine pH was determined in all animals 12, 24, and 48 h after the PGF(2alpha) injection. For Exp. 1, pH was also determined 72 and 96 h after the PGF(2alpha) injection; in Exp. 2, pH was also determined at 54, 60, 66, 72, 78, 84, 90, and 96 h after the PGF(2alpha) injection or until ovulation. A treatment x time interaction (P < 0.01) influenced concentrations of estradiol. All cows had similar (P > 0.15) concentrations of estradiol at the time of ECP administration, but after ECP treatment all cows treated with ECP and control cows that exhibited estrus had greater (P < 0.01) concentrations of estradiol compared with nontreated cows that did not exhibit estrus. In all animals, estradiol diminished 48 h after the PGF(2alpha) (time of the second GnRH injection), but ECP-treated cows, regardless of estrus, had elevated (P < 0.02) concentrations of estradiol compared with control cows. There was a treatment x time interaction (P < 0.001) on uterine pH. All cows had similar uterine pH (P > 0.19) 24 h after the PGF(2alpha) injection. Control cows that did not exhibit estrus had a greater uterine pH compared with control cows that exhibited estrus (P < 0.01) and ECP cows that exhibited estrus (P = 0.05) 48 h after the PGF(2alpha) injection (7.0 +/- 0.1 vs. 6.7 +/- 0.1 and 6.8 +/- 0.1, respectively). Estradiol cypionate-treated cows not exhibiting estrus were intermediate (6.8 +/- 0.1; P > 0.05). All cows had similar uterine pH 72 h after the PGF(2alpha) injection through ovulation (P > 0.06). In summary, uterine pH was similar among all animals that exhibited estrus, regardless of treatment with ECP.