Abstract The pharmacokinetics of moxifloxacin were investigated in buffalo calves following a single intravenous and intramuscular administration of moxifloxacin (5 mg kg −1 body wt.). Moxifloxacin concentrations in plasma and urine were determined by microbiological assay. Pharmacokinetic analysis of disposition data indicated that intravenous administration data were best described by a two compartment open model, whereas intramuscular administration data were best described by a one compartment open model. Following intravenous administration, the elimination half life ( t 1/2 β ), volume of distribution (Vd (area)) and total body clearance were 2.69 ± 0.14 h, 1.43 ± 0.08 L kg −1 and 371.2 ± 11.2 ml kg −1 h −1, respectively. Following intramuscular administration, the absorption half life ( t 1/2ka) was 0.83 ± 0.20 h. The systemic bioavailability ( F) of moxifloxacin in buffalo calves was 80.0 ± 4.08%. Urinary excretion of moxifloxacin was less than 14% after 24 h of administration of drug. In vitro binding of moxifloxacin to plasma proteins of buffalo calves was 28.4 ± 3.77%. From the data of surrogate markers (AUC/MIC, C max/MIC), it was determined in the buffalo calves that when administered by intravenous or intramuscular route at 5 mg kg −1, moxifloxacin is likely to be effective against bacterial isolates with MIC ⩽ 0.1 μg ml −1.