Acetaminophen (APAP) is one of the most intensively studied compounds that causes hepatotoxicity in the pericentral region of the liver lobules. However, spatio-temporal information on the distribution of APAP, its metabolites and GSH adducts in the liver tissue is not yet available. Here, we addressed the question, whether APAP-GSH adducts and GSH depletion show a zonated pattern and whether the distribution of APAP and its glucuronide as well as sulfate conjugates in liver lobules are zonated. For this purpose, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) technique was established, where the MSI images were superimposed onto CYP2E1 immunostained tissue. A time-dependent analysis (5, 15, 30, 60, 120, 240, 480 min) after intraperitoneal administration of 300 mg/kg APAP and a dose-dependent analysis (56 up to 500 mg APAP/kg) at 30 min were performed. The results demonstrate that the MALDI MSI technique allows the assignment of compounds and their metabolites to specific lobular zones. APAP-GSH adducts and GSH depletion occurred predominantly in the CYP2E1-positive zone of the liver, although GSH also decreased in the periportal region. In contrast, the parent compound, APAP sulfate and APAP glucuronide did not show a zonated pattern and tissue concentrations showed a similar time course as the corresponding analyses were performed with blood from the portal and liver veins. In conclusion, the present study is in agreement with the concept that pericentral CYPs form NAPQI that in the same cell binds to and depletes GSH but a lower level of GSH adducts is also observed in the periportal region. The results also provide further evidence of the recently published concept of 'aggravated loss of clearance capacity' according to which also liver tissue that survives intoxication may transiently show decreased metabolic capacity.