The role of mdr1a-encoded P-glycoprotein on transport of several fluoroquinolones across the blood–brain barrier was investigated. In vitro, P-glycoprotein substrates were selected by using a confluent monolayer of MDR1-LLC-PK1 cells. The inhibition of fluoroquinolones (100 μM) on transport of rhodamine-123 (1 μM) was compared with P-glycoprotein inhibitors verapamil (20 μM) and SDZ PSC 833 (2 μM). Subsequently, transport polarity of fluoroquinolones was studied. Sparfloxacin showed the strongest inhibition (26%) and a large polarity in transport, by P-glycoprotein activity. In vivo, using mdr1a (−/−) and wild-type mice, brain distribution of pefloxacin, norfloxacin, ciprofloxacin, fleroxacin and sparfloxacin was determined at 2, 4, and 6 h following intra-arterial infusion (50 nmol/min). Brain distribution of sparfloxacin was clearly higher in mdr1a (−/−) mice compared with wild-type mice. Sparfloxacin was infused (50 nmol/min) for 1, 2, 3 and 4 h in which intracerebral microdialysis was performed. At 4 h, in vivo recovery (dynamic-no-net-flux method) was 6.5±2.2 and 1.5±0.5%; brainECF concentrations were 5.1±0.2 and 26±21 μM; and total brain concentrations were 7.2±0.3 and 23±0.3 μM in wild-type and mdr1a (−/−) mice, respectively. Plasma concentrations were similar (18.4±0.7 and 17.9±0.5 μM, respectively). In conclusion, sparfloxacin enters the brain poorly mainly because of P-glycoprotein activity at the blood–brain barrier.