Burkholderia contaminans strain MS14 has a broad range of antifungal activities to plant and human pathogens. In previous studies, a 22.7-kb genomic fragment harboring six genes was shown to be involved in the production of an antifungal oligopeptide in B. contaminans strain MS14. In this study, another LuxR-type regulatory gene, named ambR1, was identified downstream of the ambR2 gene, and three new ORFs were found upstream of ORF6 of the 22.7-kb fragment. Site-directed mutagenesis revealed that ambR1 was required for expression of the antifungal activity against the indicator fungus Geotrichum candidum. Transcription of all the putative genes (ORFs 2-9) identified in the region except ORF1 was regulated by both ambR1 and ambR2. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1:nptII). Two of the three ORFs identified upstream from the ORF6 were predicted to encode two nonribosomal peptide synthetases (ORF7 and ORF9), and an insertion mutation in ORF9 resulted in the loss of antifungal activity against G. candidum. These results suggest that ambR1 is the key regulatory gene controlling the production of the antifungal activity of strain MS14.