The metabolism of neostigmine by fractions of rat liver has been investigated using 14C neostigmine. It has been shown that the drug is metabolized to 3-hydroxy-phenyltrimethylammonium by the microsome fraction of rat liver and that the metabolism is completely inhibited by SKF 525-A. These results support the evidence from in vivo studies that the metabolism of neostigmine is inhibited by SKF 525-A. It is suggested that the clinical effects of neostigmine may be influenced by concurrent treatment with other drugs which are metabolized by the liver. © 1968.