ABSTRACT The increasing resistance of Candida species to fluconazole is cause for concern. To determine the molecular mechanisms involved in resistance to fluconazole, I used a scheme of transposon mutagenesis in Saccharomyces cerevisiae , a genetically tractable yeast that is closely related to Candida albicans . This technique, which permits the generation and analysis of multiple random Tn 3 :: LEU2 :: lacZ fusions, can be used as a disruption mutagen (N. B. Burns et al., Genes Dev. 8:1087–1105, 1994). By using the Tn 3 :: LEU2 :: lacZ library as a disruption mutagen, I found recessive mutations in genes that were previously found to be involved in azole resistance, e.g., PDR5 and CPR1 , and in genes previously found to be involved in azole sensitivity, e.g., ERG3 . This approach also enabled me to identify recessive mutations in three genes not previously known to be involved in azole sensitivity. Two of the genes, ADA3 and SPT7 , are general transcriptional regulators; the third, YMR034c , is a putative sterol transporter. Finally, by screening the Tn 3 :: LEU2 :: lacZ library for lacZ fusions induced by a low concentration of fluconazole, I identified genes known to be induced by azoles as well as a variety of other genes not previously known to be induced by the drug. In conclusion, transposon mutagenesis is a promising screening tool for use in identifying novel drug targets and in uncovering the mechanisms involved in the response of S. cerevisiae to antifungal drugs.