Introduction Acetaminophen (paracetamol) is mainly metabolized via glucuronidation and sulphation, while the minor pathway through cytochrome P450 (CYP) 2E1 is held responsible for hepatotoxicity. In obese patients, CYP2E1 activity is reported to be induced, thereby potentially worsening the safety profile of acetaminophen. The aim of this study was to determine the pharmacokinetics of acetaminophen and its metabolites (glucuronide, sulphate, cysteine and mercapturate) in morbidly obese and non-obese patients. Methods Twenty morbidly obese patients (with a median total body weight TBW of 140.1 kg range 106–193.1 kg and body mass index BMI of 45.1 kg/m 2 40–55.2 kg/m 2 ) and eight non-obese patients (with a TBW of 69.4 kg 53.4–91.7 and BMI of 21.8 kg/m 2 19.4–27.4) received 2 g of intravenous acetaminophen. Fifteen blood samples were collected per patient. Population pharmacokinetic modelling was performed using NONMEM. Results In morbidly obese patients, the median area under the plasma concentration–time curve from 0 to 8 h (AUC 0–8h ) of acetaminophen was significantly smaller ( P  = 0.009), while the AUC 0–8h ratios of the glucuronide, sulphate and cysteine metabolites to acetaminophen were significantly higher ( P  = 0.043, 0.004 and 0.010, respectively). In the model, acetaminophen CYP2E1-mediated clearance (cysteine and mercapturate) increased with lean body weight LBW (population mean relative standard error 0.0185 L/min 15 %, P  < 0.01). Moreover, accelerated formation of the cysteine and mercapturate metabolites was found with increasing LBW ( P  < 0.001). Glucuronidation clearance (0.219 L/min 5 %) and sulphation clearance (0.0646 L/min 6 %) also increased with LBW ( P  < 0.001). Conclusion Obesity leads to lower acetaminophen concentrations and earlier and higher peak concentrations of acetaminophen cysteine and mercapturate. While a higher dose may be anticipated to achieve adequate acetaminophen concentrations, the increased CYP2E1-mediated pathway may preclude this dose adjustment.