Gentamicin concentrations, pharmacokinetic parameters, and calculated doses from enzyme multiplied immunoassay (EMI) and fluorescence polarization immunoassay (FPIA) were compared in 79 samples from 39 patients. Associations between patient factors and the differences between assay results were also assessed. Concentrations were lower when measured by EMI than by FPIA in 71 of the 79 samples (p < 0.001). Mean EMI values for elimination rate constant, volume of distribution, clearance, dose, and daily dose were 10–20% higher than mean FPIA values (p = 0.01). Dosing intervals calculated from EMI and FPIA data were different in 20 pairs of intervals and varied depending on the length of calculated interval. Univariate and multivariate analyses revealed that renal function and the presence or absence of cardiovascular disease, cimetidine, or ranitidine, and heparin were related to differences between the assay results. EMI and FPIA yielded different results for gentamicin concentrations, pharmacokinetic parameters, and calculated daily doses in the clinical setting. Such differences could result in toxic or subtherapeutic doses being administered and may be related, in part, to various patient factors.