The quantitative analysis of netilmicin in plasma, peritoneal dialysate, and urine using the fluorescence polarization immunoassay (FPIA) of the Abbott TDx system is compared with the modified high-performance liquid chromatography (HPLC) method of Peng et al., which was chosen as a reference. Using the least square method, we found that the results of the FPIA (y) correlated well with those obtained with HPLC (x). The three regression equations for the plasma, peritoneal dialysate, and urine samples, respectively, were y = 0.71x + 0.44 with r = 0.88 and n = 45; y = 0.94x + 1.22 with r = 0.93 and n = 95; and y = 0.92x + 0.70 with r = 0.93 and n = 61. The corresponding mean errors (FPIA-HPLC) with their 95% confidence intervals were -0.19 (-0.38 to -0.02), 0.69 (-0.42 to 1.81), and -0.13 (-1.13 to 0.87) microgram/ml. According to results of the Wilcoxon matched-pairs signed-ranks test, these errors did not represent a significant bias. The FPIA is thus suitable for analyzing netilmicin in the three biological fluids studied except when dialysate is contaminated with Amuchina. In this case, HPLC should be used.