Enrofloxacin is widely used in veterinary medicine and is an important alternative to treating bacterial infections, which play an important role as causes of disease and death in captive snakes. Its extralabel use in nontraditional species has been related to its excellent pharmacokinetic and antimicrobial characteristics. This can be demonstrated by its activity against gram-negative organisms implicated in serious infectious diseases of reptile species with a rapid and concentration-dependent bactericidal effect and a large volume of distribution. Pharmacokinetic parameters for enrofloxacin were investigated in seven urutu pit vipers (Bothrops alternatus), following intramuscular injections of 10 mg/kg. The plasma concentrations of enrofloxacin and its metabolite, ciprofloxacin, were measured using high-performance liquid chromatography. Blood samples were collected from the ventral coccygeal veins at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 108, and 168 hr. The kinetic behavior was characterized by a relatively slow absorption (time of maximal plasma concentration = 4.50 ± 3.45 hr) with peak plasma concentration of 4.81 ± 1.12 μg/ml. The long half-life during the terminal elimination phase (t_1/2λ = 27.91 ± 7.55 hr) of enrofloxacin after intramuscular administration, calculated in the present study, could suggest that the antibiotic is eliminated relatively slowly and/or the presence of a slow absorption in urutu pit vipers. Ciprofloxacin reached a peak plasma concentration of 0.35 μg/ml at 13.45 hr, and the fraction of enrofloxacin metabolized to ciprofloxacin was 13.06%. If enrofloxacin's minimum inhibitory concentration (MIC₉₀) values of 0.5 μg/ml were used, the ratios AUC_{e c} : MIC₉₀ (276 ± 67 hr) and Cmax_{e c} : MIC₉₀ (10 ± 2) reach the proposed threshold values (125 hr and 10, respectively) for optimized efficacy and minimized resistance development when treating infections caused by Pseudomonas. The administration of 10 mg/kg of enrofloxacin by the i.m. route should be considered to be a judicious choice in urutu pit vipers against infections caused by microorganisms with MIC values ≤0.5 μg/ml. For less susceptible bacteria, a dose increase and/or an interval reduction should be evaluated.