A single dose of florfenicol (Nuflor(®)) was administered to eight healthy adult alpacas at 20 mg/kg intramuscular (i.m.) and 40 mg/kg subcutaneous (s.c.) using a randomized, cross-over design, and 28-day washout period. Subsequently, 40 mg/kg florfenicol was injected s.c. every other day for 10 doses to evaluate long-term effects. Maximum plasma florfenicol concentrations (C(max), measured via high-performance liquid chromatography) were achieved rapidly, leading to a higher C(max) of 4.31±3.03 μg/mL following administration of 20 mg/kg i.m. than 40 mg/kg s.c. (C(max): 1.95±0.94 μg/mL). Multiple s.c. dosing at 48 h intervals achieved a C(max) of 4.48±1.28 μg/mL at steady state. The area under the curve and terminal elimination half-lives were 51.83±11.72 μg/mL·h and 17.59±11.69 h after single 2 mg/kg i.m. dose, as well as 99.78±23.58 μg/mL·h and 99.67±59.89 h following 40 mg/kg injection of florfenicol s.c., respectively. Florfenicol decreased the following hematological parameters after repeated administration between weeks 0 and 3: total protein (6.38 vs. 5.61 g/dL, P<0.0001), globulin (2.76 vs. 2.16 g/dL, P<0.0003), albumin (3.61 vs. 3.48 g/dL, P=0.0038), white blood cell count (11.89 vs. 9.66×10(3)/μL, P<0.044), and hematocrit (27.25 vs. 24.88%, P<0.0349). Significant clinical illness was observed in one alpaca. The lowest effective dose of florfenicol should thus be used in alpacas and limited to treatment of highly susceptible pathogens.