Concentrations of amphotericin B (AmB) in tissues obtained at autopsy from eight patients were measured by high-performance liquid chromatography (HPLC). The patients had received doses of 101-2,688 mg of antibiotic. Highest concentrations of the drug were found in the liver; in one patient the amount of AmB in the liver was 41% of the total dose. No evidence of metabolism of the drug was observed, and bioassay of ethanol extracts of tissue showed that the drug retained activity. Three of the patients had histologic evidence of aspergillar or candidal infection in tissues with concentrations of AmB ranging from 2.5 to 166 micrograms/g. With two patients, the concentrations of the drug in the tissues exceeded by greater than or equal to 10-fold the minimum inhibitory concentrations of the drug in isolates that had been obtained from the same tissues. Unknown factors present in tissues appear to limit the in vivo activity of AmB.