Colistin plays a key role in treatment of serious infections by Pseudomonas aeruginosa. The aims of this study were to (i) identify the pharmacokinetic/pharmacodynamic (PK/PD) index (i.e., the area under the unbound concentration-time curve to MIC ratio [fAUC/MIC], the unbound maximal concentration to MIC ratio [fC(max)/MIC], or the cumulative percentage of a 24-h period that unbound concentrations exceed the MIC [fT(>MIC)]) that best predicts colistin efficacy and (ii) determine the values for the predictive PK/PD index required to achieve various magnitudes of killing effect. Studies were conducted in a one-compartment in vitro PK/PD model for 24 h using P. aeruginosa ATCC 27853, PAO1, and the multidrug-resistant mucoid clinical isolate 19056 muc. Six intermittent dosing intervals, with a range of fC(max) colistin concentrations, and two continuous infusion regimens were examined. PK/PD indices varied from 0.06 to 18 for targeted fC(max)/MIC, 0.36 to 312 for fAUC/MIC, and 0 to 100% for fT(>MIC). A Hill-type model was fit to killing effect data, which were expressed as the log(10) ratio of the area under the CFU/ml curve for treated regimens versus control. With fC(max) values equal to or above the MIC, rapid killing was observed following the first dose; substantial regrowth occurred by 24 h with most regimens. The overall killing effect was best correlated with fAUC/MIC (R(2) = 0.931) compared to fC(max)/MIC (R(2) = 0.868) and fT(>MIC) (R(2) = 0.785). The magnitudes of fAUC/MIC required for 1- and 2-log(10) reductions in the area under the CFU/ml curve relative to growth control were 22.6 and 30.4, 27.1 and 35.7, and 5.04 and 6.81 for ATCC 27853, PAO1, and 19056 muc, respectively. The PK/PD targets identified will assist in designing optimal dosing strategies for colistin.