Pseudomonas fluorescens has the ability to produce the siderophore pyoverdine, a biotechnologically significant iron chelator, which has a wide range of potential applications, such as in agriculture (iron fertilizers) and medicine (development of antibiotics). The present work aimed to evaluate the influence of culture medium composition on the production of siderophores by P. fluorescens DSM 50090, an industrial relevant strain. It was found that the bacterium grown in minimal medium succinate (MMS) had a higher siderophore production than in King B medium. The replacement of succinate by glycerol or dextrose, in minimal medium, originated lower siderophore production. The increase of succinate concentration, the addition of amino acids or the reduction of phosphate in the culture medium did not improve siderophore production by P. fluorescens. The results obtained strongly suggest that (i) MMS is more appropriate than King B for large-scale production of siderophores; (ii) the modification of the culture medium composition, particularly the type of carbon source, influences the level of siderophore secreted; (iii) the production of siderophore by P. fluorescens seems to be a tightly regulated process; once a maximum siderophore concentration has been reached in the culture medium, the bacterium seems to be unable to produce more compound.