We report here methods for simultaneous determination of in vitro desaturase activities using stable isotope tracers or using a nontracer method in place of radiotracers. Rat microsomal β¬ 6 -and β¬ 9 -desaturase activities were assayed using standard incubation medium by monitoring the increase in product due to the reactions [U-13 C]18:2n-6 3 [U-13 C]18:3n-6 or [U-13 C]16:0 3 [U-13 C]16:1n-7, respectively. The stable isotope measurements were made by high-precision gas chromatography-combustion isotope ratio mass spectrometry. Reaction-dependent changes in product or substrate concentrations were also monitored quantitatively by conventional capillary gas chromatography with flame-ionization detection. Desaturase activities calculated from these data are consistent with tracer results. Microsomal β¬ 5 -desaturase activity was monitored by quantifying the decrease in unlabeled substrate mass using the reaction 20:3n-6 3 20:4n-6. All activities showed the expected dependencies on time, temperature, pH, and microsome and substrate concentrations and were well within the range of published activities. Activities in brain, liver, kidney, and heart microsome preparations measured with either labeled or nonlabeled substrate were not significantly different, but were highly significantly different from organ to organ. These methods demonstrate a means to measure desaturase activities conveniently without radiotracers and for reactions involving substrates which are not available in labeled form.