Measurements of isotope ratios in organic compounds at nanomolar concentrations in biological fluids require sensitive and selective gas chromatographic/mass spectrometric methods. The efficiency of sample preparation procedures must be high and gas chromatographic conditions must assure high resolution chromatography producing narrow, intense peaks. Mass spectrometric conditions must create suitable mass fragments, preferably in the high mass range. Detection, controlled by selected ion monitoring (SIM), requires the use of small mass intervals and adequate acquisition times. However, the choice of optimal conditions is limited by the need to acquire multiple data points for the eluting compound. The effects of these variables on the precision of isotope ratio measurements have been investigated to develop an optimal method for isotope ratio measurements in serum bile acids at low concentrations (0.05-8 μmol l−1). With a 25 m × 0.32 mm fused silica capillary OV-1701 column and cold on-column injection, peak widths of 10 s were obtained. Quadrupole mass spectrometry in the electron impact ionization mode (70 eV) and selected ion monitoring (SIM)(mass interval 1/16 u, acquisition time 100 ms) allowed precise (cv < 1.5%) isotope ratio measurements for chenodeoxycholic, cholic and deoxycholic acid in a single run at quantities as low as 5 pmol injected on the column. SIM switching during the run permitted isotope ratio measurements (cv < 2.2%) for an additional six bile acids, normally detected in the serum of healthy subjects.