Formation of 7 alpha-hydroxycholesterol by rat liver microsomes was quantitated using a gas chromatograph-mass spectrometer (GC/MS) operated in selected ion monitoring (SIM) mode. Microsomes from normal rat livers incubated for different periods were found to yield increased 7 alpha-hydroxycholesterol with time. This was also true when incubations contained Tween-80, but in this instance, the rate of 7 alpha-hydroxycholesterol production was lower and dependent on the concentration of Tween used. Similarly, Triton X-100, Renex-30, Kyro EOB, Cutscum, and Emulgen 911 all lowered the formation of 7 alpha-hydroxycholesterol by rat liver microsomes, whereas Triton WR-1339 stimulated its production. Analysis of data obtained from following the enzyme reaction over an extended period using an integrated Michaelis-Menten equation indicated the enzyme possesses a very significant affinity for the product (Ks greater than Kp). Similar analysis shows that Tween-80 is a noncompetitive inhibitor of the enzyme.