Environmental exposure to fungi has been associated with a variety of adverse health effects. Ergosterol, a marker of total fungal biomass, can be used to quantify exposure to fungi. Unfortunately, environmental ergosterol measurement using published GC/MS/MS methods is prone to bias introduced by sample matrix effects, resulting in potential measurement inaccuracy. We developed an isotopically labeled internal standard ((13)C ergosterol) for ergosterol quantification by GC/MS/MS, to eliminate bias due to sample matrix effects and selective losses during preparation. To produce (13)C ergosterol, we grew Saccharomyces Cerevisiae on (13)C D-glucose under aerobic conditions at room temperature. The (13)C labeled ergosterol comprised 97.1% of the ergosterol in the dry yeast preparation. Ergosterol spike-recovery from house dust samples averaged 99.3% using the isotopically labeled yeast preparation as the internal standard (I.S.). By contrast, spike-recovery averaged 42.4% when 7-dehydrocholesterol (7-DHC) was the internal standard. Analysis of indoor house dust samples from a large epidemiologic study also showed the systematic underestimation of ergosterol when analyzed using 7-DHC as compared with using the yeast preparation containing the isotopically labeled authentic compound, (13)C-ergosterol.