The characteristics of clinical and environmental isolates of Pseudomonas aeruginosa fromboth hospital and community settings were analyzed in a eukaryotic virulence model employing the AX2 and X22mutants of Dictyostelium discoideum. Thirty-one strains, including two Australian epidemic strains, of P. aeruginosa were analyzed, five from environmental sources, six from clinical sources other than cystic fibrosis (CF) patients and nineteen from CF patients’ respiratory secretions. The majority of CF isolates almost uniquely supported the growth of D. discoideum. CF isolates of P. aeruginosa were found to be less virulent than isolates from other sources.Varying degrees of inhibition of the developmental cycle ofD. discoideum when growing on CF isolates were also noted. This is the first description of P. aeruginosa isolates from clinical and environmental sources supporting the growth of D. discoideum.