The purpose of this study was to establish the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in the plasma and interstitial fluid (ISF) following subcutaneous (s.c.) administration of enrofloxacin. Ultrafiltration probes were placed in the s.c. tissue, gluteal musculature, and pleural space of five calves. Each calf received 12.5 mg/kg of enrofloxacin. Plasma and ISF samples were collected for 48 h after drug administration and analyzed by high pressure liquid chromatography. Plasma protein binding of enrofloxacin and ciprofloxacin was measured using a microcentrifugation system. Tissue probes were well tolerated and reliably produced fluid from each site. The mean ± SD plasma half‐life was 6.8 ± 1.2 and 7.3 ± 1 h for enrofloxacin and ciprofloxacin, respectively. The combined (ciprofloxacin + enrofloxacin) peak plasma concentration (C_max) was 1.52 μg/mL, and the combined area under the curve (AUC) was 25.33 μg/mL. The plasma free drug concentrations were 54% and 81% for enrofloxacin and ciprofloxacin, respectively, and free drug concentration in the tissue fluid was higher than in plasma. We concluded that C_max/MIC and AUC/MIC ratios for free drug concentrations in plasma and ISF would meet suggested ratios for a targeted MIC of 0.06 μg/mL.