Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU (-)/exoS (+) or exoU (+)/exoS (-)) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU (-)/exoS (+) isolates showed significant higher levels of the median elastolytic activity when compared to the exoU (+)/exoS (-) isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU (+) /exoS (-) genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.