Pharmacokinetic properties and tissue concentrations of enrofloxacin and ciprofloxacin were compared after intramuscular (i.m.) administrations of free and liposome-encapsulated enrofloxacin at the dose of 5 mg/kg body weight (bw). Twelve healthy adult New Zealand white rabbits were used in the experiment. Blood samples were obtained at 10, 20, 40, 60 and 90 min and 2, 4, 6, 8 and 12 h and tissue samples were collected 24 h after injection. Concentrations of drugs in serum were determined by high-performance liquid chromatography. Pharmacokinetics were best described by a two-compartment open model. Results indicated that absorption rate was slow, peak concentration was higher (P < 0.05), and the time to peak concentration (tmax ≅ 1.5 h) was significantly longer (P < 0.05) for liposome-encapsulated enrofloxacin (LEE) when compared with free enrofloxacin. Values of elimination half-life (t1/2β = 12.9 h) and mean residence time (MRT = 17.6 h) of liposome-encapsulated enrofloxacin were longer (P < 0.05) and total clearance (Cl = 0.43 l/h/kg) was lower than those of free form. Moreover, the distribution volume at steady-state (Vd(ss) = 14.4 l/kg) of enrofloxacin administered encapsulated into liposomes was significantly higher (P < 0.05) than that of free enrofloxacin (FE). The tissue levels of enrofloxacin and ciprofloxacin after LEE injection were not different (P > 0.05) from FE. In conclusion, the result of present study suggest that LEE may be a beneficial and valuable formulation in the treatment of infectious diseases caused by sensitive pathogens in animals, providing sustained drug release from injection side and prolonged therapeutic serum concentrations after i.m. administration.