In order to determine whether Pseudomonas aeruginosa alkaline protease AprA is involved in facilitating siderophore-mediated iron-acquisition from human transferrins, we measured bacterial growth, the production of siderophore and AprA, iron-acquisition from transferrins, and the proteolytic cleavage of transferrins in an alkaline minimal medium (pH 8.3) containing human transferrins as an iron source and compared these on a time scale. The growth of P. aeruginosa was found to be stimulated in proportion to the iron-saturation levels of transferrins. AprA production and the proteolytic cleavage of transferrins began concomitantly with siderophore production from the early growth phase when P. aeruginosa was actively growing and consuming most iron for growth. However, the AprA-free, but siderophore-containing, culture ultrafiltrates could also remove iron from transferrin. These results indicate that alkaline protease AprA can facilitate the siderophore-mediated iron-uptake of P. aeruginosa via the proteolytic cleavage of transferrins. However, the proteolytic cleavage by AprA is not essentially required for iron-acquisition from transferrins.