Hydrogen cyanide (HCN) is a secondary metabolite produced by many antagonistic Pseudomonas species. In the present study, the gene cluster encoding HCN synthesis in a newly isolated Pseudomonas fluorescens strain, In5, from South Greenland was investigated. Sequence analysis showed that the Greenlandic hcn gene cluster comprises a novel hcn cluster. Transposon mutagenesis of strain In5 resulted in mutants In5-2E1 and In5-1H7 with no production of HCN, and mutant In5-6B9 with reduced HCN synthesis. In mutant In5-2E1, the transposon was inserted into the hcnC gene; in mutant In5-1H7, the Tn5 insertion was found in a region upstream of a putative malate:quinone oxidoreductase gene (mqo); and in mutant In5-6B9, the transposon disrupted a probable enoyl-CoA hydratase/isomerase gene. In vitro inhibition experiments with In5 (wild type) and In5-2E1 (mutant) showed that in nitrogen-rich Luria-Bertani medium, strain In5 but not the hcn mutant In5-2E1 produced HCN and inhibited the growth of hyphae of Rhizoctonia solani and Pythium aphanidermatum . In contrast, when cultivating the strains in the carbohydrate-rich potato dextrose medium, neither of the strains produced any HCN, and thus, they were unable to inhibit hyphal growth of fungi. These experiments strongly indicate that the synthesis of HCN is highly dependent on the growth medium used.