Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.