A novel, in vitro, pharmacodynamic comparison of single and divided daily dosing regimens of aminoglycosides is described. Experiments were conducted to evaluate the impact of gentamicin and tobramycin concentration on the time required for the MICs for five Pseudomonas aeruginosa strains to revert to their original values (MIC reversion time [MRT]) following single and multiple 2-h aminoglycoside exposures to 8 and 24 mg/liter. Single 2-h aminoglycoside exposures to 8 mg/liter produced culture MRTs (gentamicin, 21.5 +/- 4.0 h; tobramycin, 22.3 +/- 2.8 h) that were significantly (P < 0.05) shorter than those measured following identical exposures to 24 mg/liter (gentamicin, 28.9 +/- 3.8 h; tobramycin, 26.8 +/- 3.1 h). However, three sequential 2-h exposures to 8 mg/liter, one exposure every 8 h, produced MRTs following the third exposure (gentamicin, 68.1 +/- 5.2 h; tobramycin, 77.8 +/- 7.8 h) that were significantly longer (P < 0.005) than those determined following three 2-h exposures to 24 mg/liter, one exposure every 24 h (gentamicin, 36.1 +/- 3.0 h; tobramycin, 34.5 +/- 3.0 h). In addition, the once-daily exposure regimen to 24 mg/liter consistently produced cultures with significantly (P < 0.005) higher aminoglycoside concentration/MIC ratios compared with those for cultures reexposed to 8 mg/liter once every 8 h. These data support the concept of once-daily aminoglycoside dosing.