The use of broad-spectrum antimicrobial therapy either by intrauterine or systemic route is commonplace in equine practice, however, much remains unknown about antibiotic efficacy in this environment. We recently demonstrated the activity of penicillin and gentamicin against common endometrial pathogens ex vivo.1 The objective of this study was to measure the concentration of gentamicin and penicillin in the uterine fluid of mares following intrauterine infusion of either a standard (PPGent) or long-acting (LA-PPGent) compounded formulation of procaine penicillin and gentamicin. We hypothesized that both drug formulations would result in therapeutic concentrations following administration, with total concentrations sustained for longer using the long-acting formulation (LA-PPGent). Ten reproductively sound mares in early estrus were administered 2378mg of procaine penicillin and 200mg of gentamicin via a single intrauterine infusion in either a standard (n=5) or a lyophilized formulation suspended in a slow-release matrix (n=5). Intrauterine fluid was collected at 0.5, 2, 4, 8, 24, 32, 48 and 72 h following infusion. A pre-weighed section of umbilical tape was introduced into the uterus through a double-guarded pipette for 30 seconds and then weighed to determine the absorbed fluid volume. Penicillin and gentamicin analyses were performed using high-performance liquid chromatography(HPLC) and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), respectively. Penicillin and gentamicin concentrations were assessed between groups and over time using a repeated measures analysis with treatment, time, and their interaction in the model statement with significance set at p<0.05. Mean intrauterine drug concentrations of PPGent peaked at 0.5 hours (Penicillin: 10,123.0 ± 4,298.0 µg/mL, Gentamicin: 3,397.3 ± 1,338.5 µg/mL), well above concentrations previously shown to inhibit bacterial growth ex vivo ([1] Von Dollen et al. J Equine Vet Sci 2019;79:121-26), and exceeded MIC for relevant organisms for 72 hours (Penicillin: 2.59 ± 6.34 µg/mL, Gentamicin: 2.14 ± 2.4 µg/mL). Mean concentrations of LA-PPG were lower at peak (Penicillin: 2,213.8 ± 967.8 µg/mL – p<0.05, Gentamicin: 1,859 ± 2,413 µg/mL) and exceeded MIC for 24h and 32h, respectively. These results support the use of procaine penicillin and gentamicin as intrauterine antimicrobial therapeutics. The long-acting formulation was not found to extend the time over MIC of antibiotics in the uterus of estrus mares. Acknowledgements: LA-PPGent was prepared by SR Veterinary Technologies LLC, Windsor CO using previously lyophilized PPGent. This research was supported by the Grayson-Jockey Club Research Foundation.