Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics compared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a collection of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene, encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2, coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analysis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate's CgPDR1 gene was used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for fluconazole resistance in transformants of the C. glabrata pdr1Delta mutant strain. Five isolates harbouring the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase. Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription factor.