<p>Acetaminophen is widely recognized for its safety as a pain reliever and fever reducer at recommended doses. However, in addition to the well-known hepatotoxic and nephrotoxic effects at overdoses recent animal studies in rats have raised the possibility that acetaminophen at a high dose of 500&#xa0;mg/kg may lead to acute impairment of the blood-brain barrier (BBB). Because species differences in hepatic and renal toxicity of acetaminophen are present, we assessed here the effect of moderate and severe overdoses of acetaminophen (300&#xa0;mg/kg and 600&#xa0;mg/kg, respectively) after intraperitoneal administration in mice on BBB permeability. Using stable isotope-labeled [¹³C₁₂]sucrose as a small molecule hydrophilic marker the brain uptake clearance K_in was measured. Our results showed no significant differences in BBB permeability between vehicle control and acetaminophen treated groups (K_in of the control group = 0.070 ± 0.025 µL min⁻¹ g⁻¹, K_in of the 300&#xa0;mg/kg group = 0.059 ± 0.017 µL min⁻¹ g⁻¹, and K_in of the 600&#xa0;mg/kg group = 0.066 ± 0.010 µL min⁻¹ g⁻¹, all values mean ± SD, <i>n</i> = 6) suggesting that even high doses of acetaminophen do not acutely compromise BBB permeability in mice. We did also not observe significant changes in tight junction proteins in brain. These findings support the notion that acetaminophen effects on the BBB may be species-specific among rodents.</p>