Study Objective. To compare the effectiveness of single daily dosing (SDD) versus conventional dosing of gentamicin when administered with penicillin to treat enterococcal infections. Design. In vitro pharmacodynamic model. Setting. Hospital laboratory. Measurements and Main Results. A 24-hour in vitro pharmacodynamic model was employed to simulate SDD and 3 times/day dosing of gentamicin, in conjunction with continuously infused penicillin, against Enterococcus faecalis. Duplicate 24-hour kill curves were generated with varying concentrations of penicillin and gentamicin alone and in combination. No difference in the rate of kill was seen between any combination of penicillin and gentamicin. Regrowth occurred only with drug combinations in which penicillin was administered continuously at the minimum inhibitory concentration. Variations in the gentamicin dosing regimen did not affect regrowth. Conclusion. In the treatment of enterococcal infections, an SDD regimen for gentamicin shows no efficacy benefit compared with conventional dosing.