Objectives were to determine the effects of intrauterine (i.u.) infusion of ceftiofur hydrochloride on uterine health and fertility of dairy cows already receiving PGF(2alpha) for estrous synchronization. Holstein cows at 44 +/- 3 d in milk (DIM) were blocked by parity and diagnosis of metritis in the first 14 DIM and of other illnesses and, within each block, randomly assigned to 1 of 2 treatments: a single i.u. infusion of 125 mg of ceftiofur hydrochloride at 44 +/- 3 DIM (ceftiofur, n = 396), or no i.u. infusion (control, n = 416). All cows received 25 mg of PGF(2alpha) at 37 +/- 3 and 51 +/- 3 DIM as part of an estrous synchronization protocol. A subset of 547 cows was evaluated for clinical endometritis immediately before treatment, and 202 cows had an aseptic uterine sample collected before the injection of PGF(2alpha) at 51 +/- 3 DIM for bacteriology and diagnosis of subclinical endometritis (> or =5% neutrophils). Pregnancy on d 38 +/- 3 and 180 +/- 7 after the first artificial insemination, pregnancy loss, and interval from calving to pregnancy in the first 300 DIM were evaluated. The proportions of cows diagnosed with clinical endometritis before treatment were similar between ceftiofur and control treatments. Intrauterine infusion with ceftiofur did not influence prevalence of subclinical endometritis and positive uterine culture 7 d after treatment; however, it reduced the prevalence of positive uterine culture in cows with clinical endometritis (29.0 vs. 51.4%) and reduced the overall prevalence of Arcanobacterium pyogenes (1.0 vs. 7.6%) at 51 +/- 3 DIM. Cows with clinical endometritis had increased prevalence of A. pyogenes (10.3 vs. 1.5%), Escherichia coli (5.9 vs. 0.75%), and overall positive uterine culture (41.2 vs. 22.4%); however, cows with subclinical endometritis only had an increased prevalence of A. pyogenes (10.2 vs. 1.5%). Ceftiofur did not affect pregnancy per artificial insemination in all cows or in cows previously diagnosed with metritis or clinical endometritis. Interval to pregnancy was similar for control and ceftiofur cows. Intrauterine infusion of ceftiofur hydrochloride reduced the prevalence of uterine infection in cows with clinical endometritis, and the prevalence of A. pyogenes, but did not affect the prevalence of subclinical endometritis or fertility of dairy cows already receiving PGF(2alpha).