The pharmacokinetics of the aminoglycoside tobramycin was evaluated after oral administration to fed or fasting (15 h) mice. As expected, under normal feeding conditions, oral absorption was negligible; however, fasting induced a dramatic increase in tobramycin bioavailability. The dual-sugar test with lactulose and l-rhamnose confirmed increased small bowel permeability via the paracellular route in fasting animals. When experiments aimed at increasing the oral bioavailability of hydrophilic compounds are performed, timing of fasting should be extremely accurate.