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Lanosterol synthase (LS) was isolated from microsomes from the plant pathogenic fungus Leptosphaeria nodorum (L. nodorum) and characterized with respect to its physical and kinetic properties. A medium throughput radiometric assay was developed for LS in order to screen potential inhibitors and to determine its kinetic properties. Quantification of LS activity relies on the separation of (±)2,3-oxidosqualene from lanosterol by reverse-phase high-pressure liquid chromatography (RP-HPLC) followed by analysis in a flow scintillation counter. LS from L. nodorum has an apparent Km (oxidosqualene) of 43 μM and an apparent native molecular weight of 150 kDa as determined by size exclusion chromatography. L. nodorum LS was compared to LS from Fusarium culmorum (F. culmorum), Fusarium graminearum (F. graminearum), and Saccharomyces cerevisiae (S. cerevisiae) for kinetic properties and inhibitory activity of a known LS inhibitor (4-bromophenyl)[2-fluoro-4-[[6-(methyl-2-propenylamino)hexyl]oxy]phenyly]-methanone (compound 1) and a derivative of this inhibitor (compound 2). While the Km (oxidosqualene) was similar for LS from all four organisms, there were differences in the activity of the two inhibitors against LS from these species. The characteristics of the assay and its use for screening molecules to find fungal inhibitors are described. To compare the biological activity of the inhibitors with their activity against LS, they were tested to determine the extent to which they suppressed the growth of L. nodorum and F. culmorum. The results suggest that the fungicidal activity of these inhibitors is related to their inhibitory effect on LS.