During once-daily dosing regimens of aminoglycosides, administration of large single doses results in high peak levels and low 24-h trough levels. However, commercial assays for monitoring aminoglycoside levels are designed to cover the smaller range of serum concentrations usually observed during multiple daily dosing regimens. The study assessed (a) the range of serum concentrations during once-daily dosing of amikacin and (b) the performance of a widely used assay system for measuring concentrations within this range. A total of 42 dosing intervals from eight patients receiving a once-daily regimen of amikacin (20 mg/kg) were monitored. Median (and range) of peak, 8- and 24-h trough levels were 61 (25-89), 5.9 (2.2-19), and 1.3 (< 0.8-6.2) mg/L, respectively. The accuracy of a fluorescence polarization immunoassay for measuring concentrations of amikacin during once-daily dosing regimens was assessed in an international multicenter study. The performance of the assay was excellent for peak and 8-h concentrations; median deviations from the target concentrations were < 5%. The majority of the trough levels (26 of 42) measured in patients during once-daily treatment were within the range of 1-2 mg/L and could also be determined with an accuracy sufficient for clinical monitoring (median deviations 14%).