The effect of single and multiple 2-h aminoglycoside exposures on the duration of the postantibiotic effect (PAE) and bacterial killing were investigated using a reference strain (ATCC 27853) and four clinical isolates of Pseudomonas aeruginosa. Concentration-dependent PAE and bacterial killing were demonstrated following single exposures to amikacin, gentamicin or tobramycin. Cultures were also repeatedly exposed to peak aminoglycoside concentration simulating traditional (three times a day) and once daily dosing regimens. Aminoglycoside re-exposures every 8 h at the original culture MIC produced significant, successive reductions in PAE (P < 0.05) and bacterial killing (P < 0.01) coincident with increases in culture MICs. Identical experiments re-exposing cultures to their predetermined, induced MICs demonstrated similar to significantly longer PAEs (P < 0.05) than those observed following first exposure. Multiple gentamicin or tobramycin exposures at 8 mg/L (traditional peak) every 8 h or 24 mg/L (once daily peak) once every 24 h both showed significant reductions in PAE (P < 0.05) and bacterial killing (P < 0.05) on repeated dosing. However, the once-daily high peak concentration exposure demonstrated significantly longer PAEs and greater bacterial killing both initially and upon re-exposure compared with the 8-hourly exposure. In addition, the once daily dosing regimen maintained substantially larger aminoglycoside concentration/MIC ratios. We conclude that multiple exposure of P. aeruginosa in-vitro, to aminoglycosides at the intervals tested, reduces the PAE and bacterial killing and increases the MICs.