The purpose of this experiment was to determine whether the time of day of single intravenous doses of gentamicin affects the drug's pharmacokinetics in dogs maintained under a 12 h light (08:00 to 20:00 h), 12 h dark (20:00 to 08:00 h) cycle. Using a crossover design, 6 mixed-breed male dogs received a single dose of 2 mg/kg of gentamicin at 8:00 or 20:00 h. Serial blood samples were collected and pharmacokinetic parameters were calculated following each timed dose. The concentration of the antibiotic was lower following the 08:00 h compared to the 20:00 h administration. When gentamicin was administered at 20:00 h, the initial concentration, mean residence time, and area under the disposition curve were significantly higher (p < 0.05) and the apparent volume of distribution of the central compartment, apparent volume of distribution, apparent volume of distribution at steady-state, and total body clearance (1.73+/-0.55 at 20:00 h versus 3.31+/-0.67 L/min/kg at 08:00 h) were significantly lower than for the 08:00 h administration (p < 0.05). Our results show that the pharmacokinetics of gentamicin exhibits significant temporal variation when administered to dogs at different times of day.