The comparative effects of ketoconazole, itraconazole, and fluconazole on the lipids of five Sporothrix schenckii strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols, by thin-layer chromatography and ultraviolet spectrophotometry. Growth of the S. schenckii isolates in the presence of azole derivative concentrations below the minimum inhibitory concentration (MIC) resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act by inhibiting ergosterol biosynthesis with a consequent accumulation of lanosterol. Concerning the MIC values, fluconazole was found to be the least effective drug. On the other hand, as determined from a comparison of the effects of the three azoles on the sterol content of the strains studied, no significant differences in efficacy were found among the tested drugs.