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Endometritis is one of the major problems in the horse breeding industry. The use of antibiotics for treatment of endometritis in the mare is recommended as best practice. The intrauterine application of antibiotics, however, has been under discussion over the last years because of concerns about its efficacy. The systemic use of antibiotics has been considered more effective because of its better distribution within the uterus. The objective of the present study was to determine the concentration of ceftiofur derivates in serum and endometrial tissue after intramuscular administration. Specifically, the authors tested the hypothesis that ceftiofur concentrations in serum and endometrial tissue remain above the minimum inhibitory concentration (MIC) for common uterine pathogens for 24 h. Nine mares in estrus received a single dose of 2.2 mg/kg ceftiofur hydrochloride intramuscular per kg of body weight. Blood samples and endometrial tissue were obtained immediately before treatment (-1 h) and 2 h and 24 h after treatment. Endometrial tissue was collected with a Kevorkian biopsy punch. Additional blood samples were collected 4 h and 10 h after treatment from the jugular veins. For determination of ceftiofur derivates in serum and endometrial tissue a high performance liquid chromatography (HPLC) assay was used. Results in serum and uterine tissue revealed greatest concentration of ceftiofur at 2 h and lowest concentrations at 24 h after treatment. Concentrations of ceftiofur at 2 and 24 h after treatment were significantly greater in serum than in endometrial tissue, but remained above the reported MIC for Streptococcus equi zooepidemicus and Escherichia coli in both serum and endometrial tissue until 24 h after treatment.