<p>[Purpose] In common methods of oral exposure of chemicals in fish such as adding to water and mixing in food, the dose depends on the spontaneous uptake by fish. So forced oral administration is applied when the dose needs to be strictly controlled, but the reports on their pharmacokinetics are few. In this study, we investigated the pharmacokinetics of acetaminophen (AAP) after forced oral administration in zebrafish which is well absorbed and metabolized in mammals. We used the administration method which is able to be applied under unanesthesia (Fundam Toxicol Sci. 8: 183-187. 2021.). </p><p>[Methods] Zebrafish were administrated AAP (50 μg/10 μL/g), then reared individually. Blood was sampled from eye socket under anaesthesia at 0.25-4 h after administration, and the concentrations of AAP and its metabolites were analyzed by LC-MS. Rearing water was collected at 1 h and the amount of AAP in water was evaluated.</p><p>[Results and discussion] AAP reached its maximum blood concentration at 0.25 h after administration and decreased to 1/20th of that concentration at 4 h. The half-life of AAP was approximately 1 h. Sulfate and glucuronide were detected and their concentrations were maximum at 4 h. In addition, 16% of dose of AAP was detected in the rearing water, which was presumably due to excretion or leakage by backflow from the oral cavity. These results indicate at least 84% of the AAP in zebrafish forced oral administrated was exposed to the body, rapidly absorbed and transferred into the blood, and then metabolized by conjugation like mammals.</p>