An isotope dilution mass spectrometric (ID/MS) method for cholesterol is described that uses capillary gas chromatography with cholesterol-13C3 as the labeled internal standard. Labeled and unlabeled cholesterol are converted to the trimethylsilyl ether. Combined capillary column gas chromatography and electron impact mass spectrometry are used to obtain the abundance ratio of the unlabeled and labeled [M+.] ions from the derivative. Quantitation is achieved by measurement of each sample between measurements of two standards whose unlabeled/labeled ratios bracket that of the sample. Seven pools were analyzed by this method: standard reference material (SRM) 1951, which consists of three frozen serum pools with low, medium, and high levels of cholesterol; SRM 1952, which consists of three freeze-dried serum pools with low, medium, and high levels of cholesterol; and SRM 909, a freeze-dried serum pool. The method is a modification of our original definitive method for cholesterol. The modified method uses much better chromatographic separations to assure specificity and a new method of implementing selected ion monitoring on a magnetic mass spectrometer to obtain high-precision measurements of ion intensity ratios on narrow gas chromatographic peaks. The modified method has a coefficient of variation (CV) of 0.22%, which is an improvement over the original method's CV of 0.36%. The measurements were found to be free of interference. The high precision and absence of bias qualify this method as a candidate definitive method.