Three fungal strains isolated from tobacco were cultured on tobacco water extracts. In these cultures, the mycelium weight was shown to be correlated with the concentration of a steroid, ergosta-5,7,22-trien-3beta-ol (ergosterol ). This steroid is not a tobacco constituent, but tobacco samples where mold or yeast infections had occured exhibited significant amounts of it. A method is proposed to quantify ergosterol in tobacco samples by reverse-phase high-performance liquid chromatography (HPLC) with UV detection at 282 nm. 7-Dehydrocholesterol can be used as internal standard. When found in a tobacco sample, the ergosterol concentration exhibits a good correlation with that of another related steroid, ergosta-4,6,8(14),22-tetraen-3-one (ETO ), for which an HPLC quantification method in tobacco is proposed. Because it is highly fluorescent, ETO is also amenable to a sensitive and quick determination by thin-layer chromatography (TLC). Once produced on tobacco, erg osterol concentration remains stable through storage under normal conditions, and even primary processing does not alter it appreciably. Possible applications of ergosterol analysis to the detection of fungal infections or the monitoring of fungal growth on tobacco are outlined. In addition, TLC estimation of the ETO concentration in a sample may constitute a convenient and fast screening method for fungal infections.