Formation of 7 alpha-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer operated in selected ion monitoring mode. This procedure measures the mass of 7 alpha-hydroxycholesterol formed and does not require addition of labeled cholesterol to monitor the reaction, since endogenous microsomal cholesterol serves as substrate for the enzyme cholesterol 7 alpha-hydroxylase. The method requires only the extraction of lipids from microsomal incubations and injection of a small fraction of this extract into the apparatus. Microsomes from normal rat livers incubated for different periods were found to yield increased 7 alpha-hydroxycholesterol with time.