Pyoverdine, the yellow-green fluorescent pigment produced by Pseudomonas aeruginosa, is a highly efficient siderophore. Pyoverdine-deficient (pvd) mutants of P. aeruginosa PAO isolated after mutagenesis were non-fluorescent and unable to grow in the presence of 2.8 mM ethylenediamine-di-(o-hydroxyphenylacetate) (EDDHA). Addition of purified pyoverdine to media containing EDDHA restored growth of pvd mutants. 6 pvd mutations were mapped between catA and mtu-9002 (at 65-70 min on the chromosome map) by R68.45-mediated conjugation. 2 slightly leaky pvd mutations were localised between argC and strA (at 35 min) by transduction. Thus, we have identified at least 2 genes or gene clusters required for pyoverdine production in P. aeruginosa.