A sensitive and specific high-performance liquid chromatographic ultraviolet method for the determination of fluconazole in human whole blood has been developed and validated. Whole blood samples were processed by a solid phase extraction procedure using an Oasis HLB extraction cartridge before chromatography. Phenacetin was used as the internal standard. Chromatography was performed using Waters C18 Symmetry analytical column, 5 microm, 4.6 x 250 mm, using an isocratic elution with a mobile phase consisting of acetonitrile and water (36:64, v/v) at a flow rate of 0.8 mL/min. The retention times of fluconazole and phenacetin were 4.7 and 8.3 minutes, respectively, and the total run time was 10 minutes. Quantitative analysis was performed using a Waters UV-VIS detector at a wavelength of 210 nm. The assay was linear over the concentration range of 0.5 to 15 microg/mL for fluconazole. The extraction recoveries at concentrations of 2.5, 5, and 10 microg/mL were 105.9%, 98.4%, and 95%, respectively. The method can quantify 0.5 microg/mL fluconazole using 300 microL of whole blood. At concentrations of 2.5, 5, and 10 microg/mL, the intraday precision expressed as coefficient of variation was 3.47%, 8.81%, and 1.14% and the interday precision was 5.21%, 5.48%, and 7.18%, respectively. This method is simple, uses a low blood volume for analysis, and allows reproducible and accurate measurement of fluconazole in whole blood samples from pediatric patients.